Apparatuses, Compositions, and Methods for Prolonging Survival of Platelets

ABSTRACT

The present invention provides modified platelets having a reduced platelet clearance and methods for reducing platelet clearance. Also provided are compositions for the preservation of platelets. The invention also provides methods for making a pharmaceutical composition containing the modified platelets and for administering the pharmaceutical composition to a mammal to mediate hemostasis.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/291,402, filed Nov. 8, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/574,857, filed Mar. 7, 2007 (371 date Dec. 31,2007), now U.S. Pat. No. 8,052,667, issued Nov. 8, 2011, which is theU.S. National stage of International Application No. PCT/US2005/031921,filed on Sep. 7, 2005, which claims the benefit of U.S. ProvisionalApplication No. 60/678,724, filed May 6, 2005, U.S. ProvisionalApplication No. 60/619,176, filed on Oct. 15, 2004, and U.S. ProvisionalApplication No. 60/607,600, filed on Sep. 7, 2004. The entire teachingsof the above applications are incorporated herein by reference.

FIELD OF THE INVENTION

The inventions relate to compositions and methods for reducing theclearance of platelets and prolonging the survival of platelets.

BACKGROUND OF THE INVENTION

Platelets are anucleate bone marrow-derived blood cells that protectinjured mammals from blood loss by adhering to sites of vascular injuryand by promoting the formation of plasma fibrin clots. Humans depletedof circulating platelets by bone marrow failure suffer from lifethreatening spontaneous bleeding, and less severe deficiencies ofplatelets contribute to bleeding complications following trauma orsurgery.

A reduction in the number of circulating platelets to below −70,000 peruL reportedly results in a prolongation of a standardized cutaneousbleeding time test, and the bleeding interval prolongs, extrapolating tonear infinity as the platelet count falls to zero. Patients withplatelet counts of less than 20,000 per uL are thought to be highlysusceptible to spontaneous hemorrhage from mucosal surfaces, especiallywhen the thrombocytopenia is caused by bone marrow failure and when theaffected patients are ravaged with sepsis or other insults. The plateletdeficiencies associated with bone marrow disorders such as a plasticanemia, acute and chronic leukemias, metastatic cancer but especiallyresulting from cancer treatment with ionizing radiation and chemotherapyrepresent a major public health problem. Thrombocytopenia associatedwith major surgery, injury and sepsis also eventuates in administrationof significant numbers of platelet transfusions.

A major advance in medical care half a century ago was the developmentof platelet transfusions to correct such platelet deficiencies, and over9 million platelet transfusions took place in the United States alone in1999 (Jacobs et al., 2001). Platelets, however, unlike all othertransplantable tissues, do not tolerate refrigeration, because theydisappear rapidly from the circulation of recipients if subjected toeven very short periods of chilling, and the cooling effect thatshortens platelet survival is irreversible (Becker et al., 1973; Bergeret al., 1998).

The resulting need to keep these cells at room temperature prior totransfusion has imposed a unique set of costly and complex logisticalrequirements for platelet storage. Because platelets are activelymetabolic at room temperature, they require constant agitation in porouscontainers to allow for release of evolved CO₂ to prevent the toxicconsequences of metabolic acidosis. Room temperature storage conditionsresult in macromolecular degradation and reduced hemostatic functions ofplatelets, a set of defects known as “the storage lesion” (Chemoff andSnyder, 1992). But the major problem with room-temperature storage,leading to its short (5-day) limitation, is the higher risk of bacterialinfection. Bacterial contamination of blood components is currently themost frequent infectious complication of blood component use, exceedingby far that of viral agents (Engelfriet et al., 2000). In the USA,3000-4500 cases yearly of bacterial sepsis occur because of bacteriallycontaminated blood components (Yomtovian et al., 1993).

The mechanism underlying the unique irreversible cold intolerance ofplatelets has been a mystery as has its physiological significance.Circulating platelets are smooth-surfaced discs that convert to complexshapes as they react to vascular injury. Over 40 years ago investigatorsnoted that discoid platelets also change shape at refrigerationtemperatures (Zucker and Borrelli, 1954). Subsequent evidence that adiscoid shape was the best predictor of viability for platelets storedat room temperature (Schlichter and Harker, 1976) led to the conclusionthat the cold-induced shape change per se was responsible for the rapidclearance of chilled platelets. Presumably irregularly-shaped plateletsdeformed by cooling became entrapped in the microcirculation.

Based on our studies linking signaling to the mechanisms leading toplatelet shape changes induced by ligands (Hartwig et al., 1995), wepredicted that chilling, by inhibiting calcium extrusion, could elevatecalcium levels to a degree consistent with the activation of the proteingelsolin, which severs actin filaments and caps barbed ends of actinfilaments. We also reasoned that a membrane lipid phase transition atlow temperatures would cluster phosphoinositides. Phosphoinositideclustering uncaps actin filament barbed ends (Janmey and Stossel, 1989)to create nucleation sites for filament elongation. We producedexperimental evidence for both mechanisms, documenting gelsolinactivation, actin filament barbed end uncapping, and actin assembly incooled platelets (Hoffmeister et al., 2001; Winokur and Hartwig, 1995).Others have reported spectroscopic changes in chilled plateletsconsistent with a membrane phase transition (Tablin et al., 1996). Thisinformation suggested a method for preserving the discoid shape ofchilled platelets, using a cell-permeable calcium chelator to inhibitthe calcium rise and cytochalasin B to prevent barbed end actinassembly. Although addition of these agents retained platelets in adiscoid shape at 4° C. (Winokur and Hartwig, 1995), such platelets alsoclear rapidly from the circulation, as we report here. Therefore, theproblem of the rapid clearance of chilled platelets remains, and methodsof increasing circulation time as well as storage time for platelets areneeded.

SUMMARY OF THE INVENTION

The present invention provides modified platelets having a reducedplatelet clearance and methods for reducing platelet clearance. Alsoprovided are compositions and methods for the preservation and storageof platelets, such as mammalian platelets, particularly human platelets.The invention also provides methods for making a pharmaceuticalcomposition containing the modified platelets and for administering thepharmaceutical composition to a mammal to mediate hemostasis.

It has now been discovered that cooling of human platelets causesclustering of the von Willebrand factor (vWf) receptor complex α subunit(GP1bα) complexes on the platelet surface. The clustering of GP1bαcomplexes on the platelet surface elicits recognition by macrophagecomplement type three receptors (αMβ2, CR3) in vitro and in vivo. CR3receptors recognize N-linked sugars with terminal βGlcNAc on the surfaceof platelets, which have formed GP1bα complexes, and phagocytose theplatelets, clearing them from the circulation and resulting in aconcomitant loss of hemostatic function.

Applicants have discovered that treatment of platelets with an effectiveamount of a glycan modifying agent such as N-acetylneuraminic acid(sialic acid), or certain nucleotide-sugar molecules, such as CMP-sialicacid or UDP-galactose leads to sialylation or glycation of the exposedβGlcNAc residues on GP1bα. Effective amounts of a glycan modifying agentrange from about 1 micromolar to about 10 millimolar, about 1 micromolarto about 1 millimolar, and most preferably about 200 micromolar to about600 micromolar of the glycan modifying agent. This has the functionaleffect of reducing platelet clearance, blocking platelet phagocytosis,increasing platelet circulation time, and increasing both plateletstorage time and tolerance for temperature changes. Additionally,platelets removed from a mammal may be stored cold for extended periods,i.e., at 4 degrees C. for 24 hours, 2 days, 3 days, 5 days, 7 days, 12days or 20 days or more, without significant loss of hemostatic functionfollowing transplantation. Cold storage provides an advantage that itinhibits the growth of contaminating microorganisms in the plateletpreparation, important as platelets are typically given to cancerpatients and other immunocompromised patients.

According to one aspect of the invention, methods for increasing thecirculation time of a population of platelets is provided. The methodcomprises contacting an isolated population of platelets with at leastone glycan modifying agent in an amount effective to reduce theclearance of the population of platelets. In some embodiments, theglycan modifying agent is selected from the group consistingUDP-galactose and UDP-galactose precursors. In some preferredembodiments, the glycan modifying agent is UDP-galactose.

In some embodiments, the method further comprises adding an enzyme thatcatalyzes the modification of a glycan moiety on the platelet. Oneexample of an enzyme that catalyzes the modification of the glycanmoiety is galactosyl transferase, particularly a beta-1-4-galactosyltransferase. Another example of an enzyme that catalyzes themodification of a glycan moiety is a sialyl transferase, which addssialic acid to the terminal galactose on the glycan moiety of theplatelet.

In one of the preferred embodiments, the glycan modifying agent isUDP-galactose and the enzyme that catalyzes the modification of theglycan moiety is galactosyl transferase. In certain aspects, the glycanmodifying agent further includes a second chemical moiety, which isadded to the glycan on the platelet in a directed manner. An example ofthis second chemical moiety is polyethylene glycol (PEG), which whencoupled to the glycan modifying agent such as UDP-galactose asUDP-galactose-PEG, in the presence of an enzyme such as galactosyltransferase, will catalyze the addition of PEG to the platelet at theterminus of the glycan moiety. Thus in certain embodiments, theinvention provides for compositions and methods for the targetedaddition of compounds to the sugars and proteins of cells.

In some embodiments, the method for increasing the circulation time of apopulation of platelets further comprises chilling the population ofplatelets prior to, concurrently with, or after contacting the plateletswith the at least one glycan modifying agent.

In some embodiments, the population of platelets retains substantiallynormal hemostatic activity.

In some embodiments, the step of contacting the population of plateletswith at least one glycan modifying agent is performed in a platelet bag.

In some embodiments, the circulation time is increased by at least about10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200%, 500% ormore.

According to another aspect of the invention, a method for increasingthe storage time of platelets is provided. The method comprisescontacting an isolated population of platelets with an amount of atleast one glycan modifying agent effective to reduce the clearance ofthe population of platelets, and storing the population of platelets.Effective amounts of a glycan modifying agent range from about 1micromolar to about 1200 micromolar, and most preferably about 200micromolar to about 600 micromolar of the glycan modifying agent. Incertain aspects the platelet preparation is stored at cold temperatures,i.e., frozen or refrigerated.

In some embodiments, the glycan modifying agent is selected from thegroup consisting of: a sugar, a monosaccharide sugar, a nucleotidesugar, sialic acid, sialic acid precursors, CMP-sialic acid,UDP-galactose, and UDP-galactose precursors. In some embodiments, theglycan modifying agent is preferably UDP-galactose.

In some embodiments, the method further comprises adding an effectiveamount of an enzyme that catalyzes the addition of the glycan modifyingagent to a glycan on the surface of the platelets. In one of thepreferred embodiments, the glycan modifying agent is UDP-galactose andthe enzyme that catalyzes the addition of the glycan modifying agent toa glycan on the surface of the platelets is galactosyl transferase,preferably a beta-1-4-galactosyl transferase. In another preferredembodiment, the glycan modifying agent is CMP-sialic acid and the enzymethat catalyzes the addition of the glycan modifying agent to a glycan onthe surface of the platelets is sialyl transferase.

In some embodiments, the method further comprises chilling thepopulation of platelets prior to, concurrently with, or after contactingthe platelets with the at least one glycan modifying agent.

In some embodiments, the population of platelets retains substantiallynormal hemostatic activity when transplanted in a mammal. Prior totransplantation the glycan modifying agent is preferably diluted orreduced to concentrations of about 50 micromolar or less.

In certain embodiments, the step of contacting the population ofplatelets with at least one glycan modifying agent is performed duringcollection of whole blood or collection of the platelets. In certainembodiments, the glycan modifying agent is introduced into a plateletbag prior to, concurrently with, or after collection of the platelets.

The platelets are capable of being stored at reduced temperatures, forexample, frozen, or chilled, and can be stored for extended periods oftime, such as at least about 3 days, at least about 5 days, at leastabout 7 days, at least about 10 days, at least about 14 days, at leastabout 21 days, or at least about 28 days.

According to another aspect of the invention, a modified platelet isprovided. The modified platelet comprises a plurality of modified glycanmolecules on the surface of the platelet. The modified glycan moleculesinclude sialic acid additions to the terminal sugar residues, orgalactosylation of the terminal sugar residues.

In some embodiments, the modified glycan molecules are moieties of GP1bαmolecules. The modified glycan molecules comprise sialic acid or atleast one added sugar molecule. The added sugar may be a natural sugaror may be a non-natural sugar. Examples of added sugars include but arenot limited to: nucleotide sugars such as UDP-galactose andUDP-galactose precursors. In one of the preferred embodiments, the addednucleotide sugar is CMP-sialic acid or UDP-galactose.

In another aspect, the invention provides a platelet compositioncomprising a plurality of modified platelets. In some embodiments, theplatelet composition further comprises a storage medium. In someembodiments, the platelet composition further comprises apharmaceutically acceptable carrier.

According to yet another aspect of the invention, a method for making apharmaceutical composition for administration to a mammal is provided.The method comprises the steps of:

(a) contacting a population of platelets contained in apharmaceutically-acceptable carrier with at least one glycan modifyingagent to form a treated platelet preparation,

(b) storing the treated platelet preparation, and

(c) warming the treated platelet preparation.

In some embodiments, the step of warming the treated plateletpreparation is performed by warming the platelets to 37° C.

In some embodiments, the step of contacting a population of plateletscontained in a pharmaceutically-acceptable carrier with at least oneglycan modifying agent comprises contacting the platelets with at leastone glycan modifying agent, alone or in the presence of an enzyme thatcatalyzes the modification of a glycan moiety. The glycan modifyingagent is preferably added at concentrations of about 1 micromolar toabout 1200 micromolar, and most preferably about 200 micromolar to about600 micromolar. In some embodiments, the method further comprisesreducing the concentration of, or removing or neutralizing the glycanmodifying agent or the enzyme in the platelet preparation. Methods ofreducing the concentration of removing or neutralizing the glycanmodifying agent or enzyme include, for example, washing the plateletpreparation or dilution of the platelet preparation. The glycanmodifying agent is preferably diluted to about 50 micromolar or lessprior to transplantation of the platelets into a human subject.

Examples of glycan modifying agents are listed above. In one of thepreferred embodiments, the glycan modifying agent is CMP-sialic acid orUDP-galactose. In some embodiments, the method further comprises addingan exogenous enzyme that catalyzes the addition of the glycan modifyingagent to a glycan moiety, such as a beta-1-4 galactosyl transferase.

In one of the preferred embodiments, the glycan modifying agent isUDP-galactose and the enzyme is galactosyl transferase.

In some embodiments, the population of platelets demonstratesubstantially normal hemostatic activity, preferably aftertransplantation into a mammal.

In certain embodiments, the step of contacting the population ofplatelets with at least one glycan modifying agent is performed duringthe collection process on whole blood or fractionated blood, such as onplatelets in a platelet bag.

In some embodiments, the platelet preparation is stored at a temperatureof less than about 15° C., preferably less than 10° C., and morepreferably less than 5° C. In some other embodiments, the plateletpreparation is stored at room temperature. In other embodiments, theplatelets are frozen, e.g., 0° C., −20° C., or −80° C. or cooler.

According to yet another aspect of the invention, a method for mediatinghemostasis in a mammal is provided. The method comprises administering aplurality of modified platelets or a modified platelet composition tothe mammal. The platelets are modified with the glycan modifying agentprior to administration, such as during collection, prior to storing,after storage and during warming, or immediately prior totransplantation.

According to still yet another aspect of the invention, a storagecomposition for preserving platelets is provided. The compositioncomprises at least one glycan modifying agent, added to the platelets inan amount sufficient to modify platelets glycans, thereby increase thestorage time and/or the circulation time of platelets added to thestorage composition by reducing platelet clearance.

In some embodiments the composition further comprises an enzyme thatcatalyzes the modification of a glycan moiety. The enzyme may beexogenously added. A beta-1-4 galatosyl transferase or a sialyltransferase, or both, exemplify preferred enzymes for catalyzing themodification of the glycan moieties on the platelets.

According to another aspect of the invention, a container for collecting(and optionally processing) platelets is provided. The containercomprises at least one glycan modifying agent in an amount sufficient tomodify glycans of platelets contained therein. The container ispreferably a platelet bag, or other blood collection device.

In some embodiments, the container further comprises an enzyme thatcatalyzes the modification of a glycan moiety with the glycan modifyingagent, such as a beta-1-4 galatosyl transferase or a sialyl transferase.

In some embodiments the container further comprises a plurality ofplatelets or plasma comprising a plurality of platelets.

In some embodiments, the glycan modifying agent is present at aconcentration higher than it is found in naturally occurring plateletsor in serum. In certain aspects these concentrations are 1 micromolar to1200 micromolar, and most preferably about 200 micromolar to about 600micromolar. In other embodiments, the beta-1-4 galatosyl transferase ora sialyl transferase is at a concentration higher than it is found innaturally occurring platelets or in serum, such as concentrations thatwould be observed if the enzyme were added exogenously to the platelets.

According to still yet another aspect of the invention, a device forcollecting and processing platelets is provided. The device comprises: acontainer for collecting platelets; at least one satellite container influid communication with said container; and at least one glycanmodifying agent in the satellite container. The container optionallyincludes an enzyme such as a beta-1-4 galatosyl transferase or a sialyltransferase.

In some embodiments, the glycan modifying agent in the satellitecontainer is present in sufficient amounts to preserve the platelets inthe container, for example from concentrations of about 1 micromolar toabout 1200 micromolar.

In some embodiments, the glycan modifying agent in the satellitecontainer is prevented from flowing into the container by a breakableseal.

In other aspects, the invention includes a kit having a sterilecontainer capable of receiving and containing a population of platelets,the container substantially closed to the environment, and a sterilequantity of a glycan modifying agent sufficient to modify a volume ofplatelets collected and stored in the container, the kit furtherincludes suitable packaging materials and instructions for use. Glycanmodifying agents in the kit include CMP-sialic acid, UDP-galactose, orsialic acid. The container is suitable for cold-storage of platelets.

The invention also includes, in certain aspects, a method of modifying aglycoprotein comprising, obtaining a plurality of platelets having GP1bαmolecules, and contacting the platelets with a glycan modifying agent,wherein the glycan modifying agent galactosylates or sialylates theterminus of a GP1bα molecule on the platelets.

The invention further includes a method of modifying a blood constituentcomprising, obtaining a sample of blood having platelets, and contactingat least the platelets with a glycan modifying agent, wherein the glycanmodifying agent galactosylates or sialylates the terminus of a GP1bαmolecule on the platelets.

In other aspects, the invention includes a method of reducing pathogengrowth in a blood sample comprising, obtaining a sample of blood havingplatelets, contacting at least the platelets with a glycan modifyingagent, wherein the glycan modifying agent galactosylates or sialylatesthe terminus of a GP1bα molecule on the platelets, and storing the bloodsample having modified platelets at a temperature of about 2 degrees C.to about 18 degrees C. for at least three days, thereby reducingpathogen growth in the blood sample.

In another aspect, the invention provides an apparatus for processing asample of blood cells, including a sterile first container having one ormore ports and containing a preparation of blood cells, a second sterilecontainer having one or more ports and containing a blood cell modifyingagent, (also referred to as a platelet solution or a glycan modifyingagent) the first container adapted to the second container through asterile conduit reversibly attachable to the first container port andthe second container port, the conduit further comprising a valve,wherein the blood cell modifying agent is introduced into the firstcontainer and the preparation of blood cells therein is rendered coldstorage competent after the blood cells are contacted with the bloodcell modifying agent. In one embodiment, the invention includes asterile third container having one or more ports adapted to the firstcontainer through a second sterile conduit reversibly attachable to thefirst container port and the third container port, the conduit furthercomprising a valve. In another embodiment, the invention includes aleukocyte filter. In various embodiments, some shown in the figures, thefirst container, second container or the third container are blood bagsor a syringe. In other embodiments, the blood cell modifying agent is anucleoside sugar such as UDP galactose, or cytidine5′monophospho-N-acetylneuraminic acid. The blood cells suitable formodification in the bioprocess include a population of plateletsobtained from individual random donor blood, pooled random donor blood,or single donor blood. In various other embodiments, the conduit isadapted to an in-line filter having a median pore diameter small enoughto substantially prevent the flow of bacteria through the in-linefilter. Preferred median pore diameters for the in-line filter are lessthan about 1 micron, more preferably less than about 0.50 microns andmost preferably about 0.22 microns. In yet another embodiment, thesecond container port has a frangible barrier. In even anotherembodiment, the first conduit or the second conduit reversibly attachesto the first container port, the second container port or the thirdcontainer port through a sterile dock.

In another aspect, the invention provides an apparatus for processing asample of blood cells, including a sterile first container having one ormore ports, and an array having a conduit and a plurality of steriledocks, wherein each of the sterile docks are reversible adaptable toblood storage containers, the blood storage containers having a sampleof blood cells and further comprising at least one port for connectingto the sterile docks of the array, wherein the blood cells areintroduced into the sterile first container through the conduit and arerendered cold storage competent after the blood cells are contacted witha blood cell modifying agent introduced into the first container. Insome embodiments, the blood cell modifying agent is a sterile nucleosidesugar such as UDP galactose or a sterile preparation of cytidine5′monophospho-N-acetylneuraminic acid. In various other embodiments, theinvention provides an in-line filter having a median pore diameter smallenough to substantially prevent the flow of bacteria through the in-linefilter. Preferred median pore diameters for the in-line filter are lessthan about 1 micron, more preferably less than about 0.50 microns andmost preferably about 0.22 microns. In one embodiment, the blood cellsfurther comprise a population of platelets obtained from individualrandom donor blood, pooled random donor blood, or single donor blood. Inanother embodiment, the array further comprises a leukocyte filterproximal to the first container. In even another embodiment, the bloodcell modifying agent is contained in the first container. In anotherembodiment, the invention includes a second container having one or moreports and containing a blood cell modifying agent, the first containeradapted to the second container through a sterile conduit reversiblyattachable to the first container port and the second container port. Instill yet another embodiment, the second container is a syringe. In oneembodiment, the conduit is adapted to an in-line filter having a medianpore diameter small enough to substantially prevent the flow of bacteriathrough the in-line filter. In another embodiment median pore diametersfor the in-line filter are less than about 1 micron, more preferablyless than about 0.50 microns and most preferably about 0.22 microns. Inanother embodiment, the second container port has a frangible barrier.

In another aspect, the invention provides an apparatus for processing asample of blood cells, including a sterile first container having one ormore ports the first container further comprising a subcontainerdisposed therein, the subcontainer having a port and a frangible barrierand containing a blood cell modifying agent, and an array comprising aconduit and a plurality of sterile docks, wherein each of the steriledocks are reversible adaptable to blood storage containers, the bloodstorage containers having a sample of blood cells and further comprisingat least one port for connecting to the sterile docks of the array,wherein the blood cells are introduced into the sterile first containerthrough the conduit and are rendered cold storage competent after theblood cells are contacted with a blood cell modifying agent introducedinto the first container. In one embodiment, the blood cell modifyingagent is a sterile nucleoside sugar such as UDP galactose or a sterilepreparation of cytidine 5′monophospho-N-acetylneuraminic acid. Inanother embodiment median pore diameters for the in-line filter are lessthan about 1 micron, more preferably less than about 0.50 microns andmost preferably about 0.22 microns. In another embodiment, the secondcontainer port has a frangible barrier. In another embodiment, the bloodcells further comprise a population of individual random donor blood,pooled random donor blood, or single donor blood. In another embodiment,the array further comprises a leukocyte filter proximal to the firstcontainer.

In one aspect, the invention provides a method for treating a bloodcell, including obtaining an apparatus as described, obtaining a sampleof blood cells including a subpopulation of platelets, and exposing theblood cells to the blood cell modifying agent in the apparatus therebyrendering the subpopulation of platelets cold-storage competent. In oneembodiment, the method includes separating the leukocytes from the bloodcells prior to exposing the blood cells to the blood cell modifyingagent. In one embodiment, the blood cell modifying agent is a sterilenucleoside sugar such as UDP galactose or a sterile preparation ofcytidine to 5′monophospho-N-acetylneuraminic acid. In another embodimentmedian pore diameters for the in-line filter are less than about 1micron, more preferably less than about 0.50 microns and most preferablyabout 0.22 microns. In another embodiment, the second container port hasa frangible barrier. In another embodiment, the blood cells furthercomprise a population of individual random donor blood, pooled randomdonor blood, or single donor blood. In another embodiment, the methodprovides that the blood cells are contacted with the blood cellmodifying agent before infusion of the treated blood cells into apatient. In another embodiment, the method provides that the blood cellsare contacted with the blood cell modifying agent before cold storage ofthe blood cells. In another embodiment, the method provides that theblood cells are contacted with the blood cell modifying agent at thetime of blood collection from a blood donor. In another embodiment, themethod provides for separating the blood cells into subpopulations ofplatelets, plasma, red blood cells and white blood cells. In anotherembodiment, the blood cells are contacted with the blood cell modifyingagent after the blood cells have been separated by apheresis.

In another aspect, the invention provides for a treated blood cellobtained through the methods described. The treated blood cells,following cold storage, are suitable for transfusion into a patient.These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent in reference to the followingdetailed description of the invention. Each of the limitations of theinvention can encompass various embodiments of the invention. It istherefore, anticipated that each of the limitations involving any oneelement or combination of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows circulation time in mice of room temperature platelets andof platelets chilled and rewarmed in the presence or absence of EGTA-AMand Cytochalasin B. The curves depict the survival of5-chloromethylfluorescein diacetate (CMFDA) labeled, room temperature(RT) platelets, platelets chilled at ice-bath temperature (Cold) andrewarmed to room temperature before injection and chilled and rewarmedplatelets treated with EGTA-AM and cytochalasin B (Cold+CytoB/EGTA) topreserve their discoid shape. Each curve represents the mean±SD of 6mice. Identical clearance patterns were observed with ¹¹¹Indium-labeledplatelets.

FIG. 1B shows that chilled platelets aggregate normally in vitro.Washed, chilled-rewarmed (Cold) or room temperature (RT) wild typeplatelets were stimulated by the addition of the indicated agonists at37° C. and light transmission was recorded on a standard toaggregometer. Aggregation responses of chilled platelets treated withEGTA-AM and cytochalasin B were identical to untreated chilledplatelets.

FIG. 1C shows that cold induced clearance occurs predominantly in theliver of mice. The liver is the primary clearance organ of chilledplatelets, containing 60-90% of injected platelets. In contrast, RTplatelets are cleared more slowly in the spleen. ¹¹¹Indium labeledplatelets were injected into syngeneic mice and tissues were harvestedat 0.5, 1 and 24 hours. Data are expressed per gram of tissue. Each bardepicts the mean values of 4 animals analyzed±SD.

FIG. 1D shows that chilled platelets co-localize with hepatic sinusoidalmacrophages (Kupffer cells). This representative confocal-micrographshows the hepatic distribution of CMFDA-labeled, chilled-rewarmedplatelets (green) after 1 hour of transfusion, which preferentiallyaccumulate in periportal and midzonal fields of liver lobules. Kupffercells were visualized after injection of nile red-labeled spheres. Themerged micrograph that shows co-localization of chilled platelets andmacrophages in yellow. The lobule organization is indicated (CV: centralvein; PV: portal vein, bar: 100 μM).

FIG. 2 shows that chilled platelets circulate normally in CR3-deficientmice, but not in complement 3 (C3) or vWf deficient mice. CMFDA-labeledchilled-rewarmed (Cold) and room temperature (RT) wild type plateletswere transfused into six each of syngeneic wild type (WT), CR3-deficient(A), vWf-deficient (B) and C3-deficient (C) recipient mice and theirsurvival times determined. Chilled platelets circulate in CR3-deficientanimals with the same kinetics as room-temperature platelets, but arecleared rapidly from the circulation of C3- or vWf-deficient mice. Dataare mean±SD for 6 mice.

FIG. 3A-3C show that chilled platelets adhere tightly to CR3-expressingmouse macrophages in vivo. FIG. 3A—Chilled-rewarmed TRITC-labeledplatelets (left panel) adhere with a 3-4× higher frequency to liversinusoids than room temperature CMFDA-labeled platelets (right panel).The intravital fluorescence micrographs were obtained 30 min after theinfusion of the platelets. FIG. 3B-Chilled-rewarmed (Cold, open bars)and room temperature platelets (RT, filled bars) adhere to sinusoidalregions with high macrophage density (midzonal) with similardistributions in wild type mice. FIG. 3C—Chilled-rewarmed plateletsadhere 3-4× more than room temperature platelets to macrophages in thewild type liver (open bars). In contrast, chilled-rewarmed or roomtemperature platelets have identical adherence to macrophages inCR3-deficient mice (filled bars). 9 experiments with wild type mice and4 experiments with CR3-deficient mice are shown (mean±SEM, *P<0.05:**P<0.01).

FIG. 4A-4C show that GP1bα mediates chilled platelet clearance,aggregates in the cold, but to binds activated vWf normally on chilledplatelets. FIG. 4A—CMFDA-labeled platelets enzymatically cleared of theGP1bα extracellular domain (left panel, inset, filled area) or controlplatelets were kept at room temperature (left panel) or chilled-rewarmed(right panel) infused into syngeneic wild type mice, and plateletsurvivals were determined. Each survival curve represents the meanvalues 3 SD for 6 mice. FIG. 4B—Chilled, or RT platelet rich plasma wastreated with (shaded area) or without (open area) botrocetin. vWf boundwas detected using FITC labeled anti-vWf antibody. FIG. 4C—The vWfreceptor redistributes from linear arrays (RT) into aggregates (Chilled)on the surface of chilled murine platelets. Fixed, chilled-rewarmed, orroom temperature platelets (RT) were incubated with monoclonal ratanti-mouse GP1bα antibodies followed by 10 nm colloidal gold particlescoated with goat anti-rat IgG. The bars are 100 nm. Inset: lowmagnification of platelets.

FIG. 5A-5C show GP1bα-CR3 interaction mediates phagocytosis of chilledhuman platelets in vitro. FIGS. 5A and 5B show a representative assayresult of THP-1 cells incubated with room temperature (RT) (FIG. 5A) orchilled-rewarmed (Cold) platelets (FIG. 5B). CM-Orange-labeled plateletsassociated with macrophages shift in orange fluorescence up the y axis.The mean percentage of the CM-Orange positive native macrophagesincubated with platelets kept at room temperature was normalized to 1.Chilling of platelets increases this shift from ˜4% to 20%. Theplatelets are predominantly ingested, because they do not dual labelwith the FITC-conjugated mAb to CD61. FIG. 5C Undifferentiated (openbars) THP-1 cells express ˜50% less CR3, and ingest half as manychilled-rewarmed platelets. Differentiation (filled bars) of CR3expression however, had no significant effect on the uptake of RTplatelets. Treatment of human platelets with the snake venommetalloprotease, mocarhagin (Moc), which removes the N-terminus of GP1bαfrom the surface of human platelets (inset; control: solid line,mocarhagin treated platelets: shaded area), reduced phagocytosis ofchilled platelets by ˜98%. Data shown are means±SD of 5 experiments.

FIG. 6A-6E show circulating, chilled platelets have hemostatic functionin CR3 deficient mice. Normal in vivo function of room temperature (RT)platelets transfused into wild type mice (FIGS. 6A and 6B) and ofchilled (Cold) platelets transfused into CR3 deficient mice (FIGS. 6Cand 6D), as determined by their equivalent presence in plateletaggregates emerging from the wound 24 hrs after infusion of autologousCMFDA labeled platelets. Peripheral blood (FIGS. 6A and 6C) and theblood emerging from the wound (shed blood, FIGS. 6B and 6D) wereanalyzed by whole blood flow cytometry. Platelets were identified byforward light scatter characteristics and binding of the PE-conjugatedanti-GP1bα mAb (pOp4). The infused platelets (dots) were identified bytheir CMFDA fluorescence and the non-infused platelets (contour lines)by their to lack of CMFDA fluorescence. In the peripheral whole bloodsamples, analysis regions were plotted around the GP1bα-positiveparticles to include 95% of the population on the forward scatter axis(region 1) and the 5% of particles appearing above this forward lightscatter threshold were defined as aggregates (region 2). The percentagesrefer to the number of aggregates formed by CMFDA-positive platelets.This shown result is representative of 4 experiments. FIG. 6E shows exvive function of CM-Orange, room temperature (RT) platelets transfusedinto wild type mice and CM-Orange, chilled-rewarmed (Cold) plateletstransfused into CR3 deficient mice, as determined by exposure ofP-selectin and fibrinogen binding following thrombin (1 U/ml) activationof blood drawn from the mice after 24 hours post infusion. CM-Orangelabeled platelets have a circulation half-life time comparable to thatof CMFDA labeled platelets (not shown). Transfused platelets wereidentified by their CM-Orange fluorescence (filled bars). Non-transfused(non-labeled) analyzed platelets are represented as open bars. Resultsare expressed as the percentage of cells present in the P-selectin andfibrinogen positive regions (region 2). Data are mean±SD for 4 mice.

FIG. 7 is a schematic depicting two platelet clearance pathways.Platelets traverse central and peripheral circulations, undergoingreversible priming at lower temperatures at the body surface. Repeatedpriming leads to irreversible GP1b-X-V (vWfR) receptor complexreconfiguration and clearance by complement receptor type 3 (CR3)bearing hepatic macrophages. Platelets are also cleared after theyparticipate in microvascular coagulation.

FIG. 8A-8D show the effect of monosaccharides on phagocytosis of chilledplatelets.

FIG. 9A-9F show the dot plots of binding of WGA lectin to roomtemperature platelets or chilled platelets.

FIG. 10 shows the analysis of various FITC labeled lectins bond to roomtemperature or chilled platelets.

FIG. 11A shows the summary of FITC-WGA binding to the surface of roomtemperature or chilled platelets obtained by flow cytometry before andafter 3-hexosaminidase treatment.

FIG. 11B shows that GP1bα removal from the platelet surface reducedFITC-WGA binding to chilled platelets.

FIG. 12 shows that galactose transfer onto platelet oligosaccharidesreduces chilled platelet (Cold) phagocytosis, but does not affect thephagocytosis of room temperature (RT) platelets.

FIG. 13 shows the survival of chilled, galactosylated murine plateletsrelative to untreated platelets.

FIG. 14A-14C show that platelets containing galactose transferases ontheir surface transfer galactose without the addition of externaltransferases as judged by WGA binding (FIG. 14A) and in vitrophagocytosis results for human platelets (FIG. 14B). FIG. 14C shows thatof UDP-galactose with or without Galactose transferase (GalT) onsurvival of murine platelets. UDP-galactose with or without GalT wasadded to murine platelets before chilling for 30 min at 37° C. Theplatelets were chilled for 2 hours in an ice bath and then transfused(10 platelets/mouse) into mice and their survival determined.

FIG. 15 shows the time course of ¹⁴C-labeled UDP-galactose incorporationinto human platelets.

FIG. 16 shows galactosylation of platelets in four platelet concentratesamples at different concentrations of UDP-galactose.

FIG. 17 shows the complement receptor mediates phagocytosis andclearance of chilled platelets.

FIG. 18 shows the GP1bα subunit of platelet von Willebrand factorreceptor binds the I-domain of αM of αM/β2 integrin.

FIG. 19 shows that chilled platelets circulate and function normally inαM knockout mice.

FIG. 20 illustrates vWf receptor inactivation.

FIG. 21 shows that αM/β2 recognizes the outer tip of GP1bα and mediatesclearance of chilled platelets, thus demonstrating that GP1bα hascoagulant (vWf binding) and non-coagulant (clearance) functions.

FIG. 22 illustrates the primary structure of αM (CD11b).

FIG. 23 shows that αM has a lectin affinity site.

FIG. 24 shows that the lectin domain of macrophage ααM/β2 receptorsrecognizes βGlcNAc residues on clustered GP1bα.

FIG. 25 shows that a soluble αM-lectin domain inhibits chilled humanplatelet phagocytosis by macrophages.

FIG. 26 shows the construction of CHO cells expressing αMαX chimericproteins.

FIG. 27 illustrates a phagocytic assay for altered platelet surfaceinduced by chilling.

FIG. 28 shows that the αM-lectin domain mediates chilled human plateletphagocytosis.

FIG. 29 shows that macrophage αM/β32 receptors recognize βGlcNAcresidues on clustered GP1bα receptors of chilled platelets.

FIG. 30 illustrates the galactosylation of platelets through GP1bα.

FIG. 31 shows expression of β4GalT1 on the platelet surface.

FIG. 32 illustrates that galatosylated chilled murine platelets cancirculate in vivo.

FIG. 33 illustrates that galatosylated chilled murine platelets canfunction normally in murine models.

FIG. 34 shows that human platelet concentrates can be galactosylated,which preserves platelet function.

FIG. 35 illustrates a method for galactosylation of human plateletconcentrates.

FIG. 36 shows surface galactose on platelet concentrates is stable.

FIG. 37 shows that galactosylation inhibits phagocytosis by THP-1macrophages of human chilled platelets.

FIG. 38 shows that platelet counts and pH remain unchanged inrefrigerated platelet concentrates.

FIG. 39 shows the effects of refrigeration and galactosylation onretention of platelet responses to agonists during storage ofconcentrates.

FIG. 40 shows the effect of storage conditions on shape change(spreading) and clumping of platelets in concentrates.

FIG. 41 illustrates an embodiment of the invention wherein a bioprocessfor collecting, treating and storing platelets is described. Plateletsare derived from a variety of blood sources, including IRDP—IndividualRandom Donor Platelets, PRDP—Pooled Random Donor Platelets andSDP—Single Donor Platelets. The container having the glycan modifyingagent, e.g., a solution of UDP-Gal and/or CMP-NeuAc is sterile docked tothe bag containing the platelets. A sterile dock is also referred to asa sterile connection device (SCD) or a total containment device (TCD).The sterile dock permits connection of two pieces of conduit whilemaintaining sterility of the system. The glycan modifying agent is mixedwith the platelets and then the modified platelets are transferred to anon-breathable bag. The glycan modifying agent can be introduced to theplatelets at a variety of times, e.g., before infusion, before storage,after componentization or directly to whole blood, or during theplatelet apheresis procedure at the time of donation. Likewise, theglycan modifying solution may be provided in a variety of forms, such asfull strength concentration liquid, concentrated liquid—diluted beforeuse, dehydrated, freeze dried, lyophilisized, powder, frozen, viscousfluid, suspension, base and activator, or reactant and catalyst. In thisembodiment, the blood is passed through a leukocyte filter. Variousmethods of leukocyte depletion are known in the art, e.g., glass wool orother affinity separation methods for removing leukocyte fractions fromwhole blood, and provide examples of means for filtering the leukocytesfrom the rest of the blood and specifically the platelets.

FIG. 42 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.This illustration is similar to FIG. 41 but does not include a leukocytefilter.

FIG. 43 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The bag containing the platelets is sterile docked to the bag containingthe platelet solution. The glycans modifying solution, also called aplatelet solution, is mixed with the platelets and then transferred to anon-breathable bag and thru a leukocyte filter.

FIG. 44 illustrates a variation of FIG. 43, that does not include aleukocyte filter.

FIG. 45 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The syringe containing the platelet solution (UDP-Gal and/or CMP-NeuAc)is sterile docked to the bag containing the platelets. The plateletsolution is mixed with the platelets and then transferred to anon-breathable bag and thru a leukocyte filter.

FIG. 46 illustrates a variation of FIG. 45, that does not include aleukocyte filter.

FIG. 47 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The bag containing the platelet solution (UDP-Gal and/or CMP-NeuAc) isconnected to the container port using a bag spike thru a 0.22 micronfilter to the bag containing the platelets. The platelet solution ismixed with the platelets and then transferred to a non-breathable bagand thru a leukocyte filter. A 0.22 micron filter is illustrated, butlarger pore diameter filters are suitable to provide increased flowrate. Median pore sizes greater than about 1 micron are not suitable forsterile filtration. Preferred sizes are less than about 0.75 microns,more preferably less than about 0.5 microns, and most preferably about0.22 microns.

FIG. 48 illustrates a variation of FIG. 47, that does not include aleukocyte filter.

FIG. 49 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The bag containing the platelet solution (either single dose or bulk) isconnected using a luer lock thru a 0.22 micron filter to the bagcontaining the platelets. The platelet solution is mixed with theplatelets and then transferred to a non-breathable bag and thru aleukocyte filter.

FIG. 50 illustrates a variation of FIG. 49, that does not include aleukocyte filter.

FIG. 51 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The syringe containing the platelet solution is connected using a luerlock thru a 0.22 micron filter to the bag containing the platelets. Theplatelet solution is mixed with the platelets and then transferred to anon-breathable bag and thru a leukocyte filter. Also shown, IRDP can bepooled to form PRDP.

FIG. 52 illustrates a variation of FIG. 51, that does not include aleukocyte filter.

FIG. 53 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The syringe containing the platelet solution is connected using a luerlock thru a 0.22 micron filter to the bag containing the platelets. Theplatelet solution is mixed with the platelets and then transferred to anon-breathable bag and thru a leukocyte filter. The syringe can beaseptically refilled from the bulk platelet solution because of thein-line filtration device.

FIG. 54 illustrates a variation of FIG. 53, that does not include aleukocyte filter.

FIG. 55 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The large non-breathable bag (final storage bag) containing the plateletsolution includes an array comprising long piece of conduit and aplurality of ports to allow the sterile docking of multiple IRDP bagssequentially from the distal end of the tube (denoted #8) to theproximal end (denoted #1) thru a 0.22 micron filter to the bagcontaining the platelet solution. The platelet solution is mixed withthe pooled platelets.

FIG. 56 illustrates a variation of FIG. 55, that does not include aleukocyte filter.

FIG. 57 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.Platelet solution delivery to the containment bag is facilitated by anSCD on the bag.

FIG. 58 illustrates a variation of FIG. 57, that does not include aleukocyte filter.

FIG. 59 illustrates a variation of FIG. 57. The large non-breathable bag(final storage bag) has the platelet solution, stored in a syringe,aseptically connected and added thru a 0.22 micron filter.

FIG. 60 illustrates a variation of FIG. 59, that does not include aleukocyte filter.

FIG. 61 illustrates a variation of the invention, wherein a containerhaving platelet solution is adapted to the container having blood cellsthrough a conduit attachable via a luer lock connection. The conduit hasa bag spike to puncture a barrier in the container, thereby permittingwithdrawal of the glycans modifying solution.

FIG. 62 illustrates a variation of FIG. 61, that does not include aleukocyte filter.

FIG. 63 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The large non-breathable bag (final storage bag) has the plateletsolution, stored in a bag, connected with a frangible plug that can beopened to deliver the platelet solution.

FIG. 64 illustrates a variation of FIG. 63, that does not include aleukocyte filter.

FIG. 65 illustrates another embodiment of the invention wherein abioprocess for collecting, treating and storing platelets is described.The large non-breathable bag (final storage bag) includes an integratedbag of platelet solution having a frangible plug that can be opened todeliver the platelet solution directly into the platelet storagecontainer.

FIG. 66 illustrates a variation of the embodiment illustrated as FIG.65. The bag having the platelet modifying solution is integrated withinthe storage bag. The platelet solution is released upon breaking of thefrangible plug or separation membrane.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a population of modified platelets that haveenhanced circulation properties and that retain substantially normal invive hemostatic activity. Hemostatic activity refers broadly to theability of a population of platelets to mediate bleeding cessation.Various assays are available for determining platelet hemostaticactivity (Bennett, J. S. and Shattil, S. J., 1990, “Platelet function,”Hematology, Williams, W. J., et al., Eds. McGraw Hill, pp 1233-12250).However, demonstration of “hemostasis” or “hemostatic activity”ultimately requires a demonstration that platelets infused into athrombocytopenic or thrombopathic (i.e., non-functional platelets)animal or human circulate and stop natural or experimentally-inducedbleeding.

Short of such a demonstration, laboratories use in vitro tests assurrogates for determining hemostatic activity. These tests, whichinclude assays of aggregation, secretion, platelet morphology andmetabolic changes, measure a wide variety of platelet functionalresponses to activation. It is generally accepted in the art that the invitro tests are reasonably indicative of hemostatic function in vivo.

Substantially normal hemostatic activity refers to an amount ofhemostatic activity seen in the modified platelets, that is functionallyequivalent to or substantially similar to the hemostatic activity ofuntreated platelets in vivo, in a healthy (non-thrombocytopenic ornon-thrombopathic mammal) or functionally equivalent to or substantiallysimilar to the hemostatic activity of a freshly isolated population ofplatelets in vitro.

The instant invention provides methods for reduced temperature storageof platelets which increases the storage time of the platelets, as wellas methods for reducing clearance of or increasing circulation time of apopulation of platelets in a mammal. Also provided are plateletcompositions methods and compositions for the preservation of plateletswith preserved hemostatic activity as well as methods for making apharmaceutical composition containing the preserved platelets and foradministering the pharmaceutical composition to a mammal to mediatehemostasis. Also provided are kits for treating a platelet preparationfor storage, and containers for storing the same.

In one aspect of the invention, the method for increasing circulationtime of an isolated population of platelets involves contacting anisolated population of platelets with at least one glycan modifyingagent in an amount effective to reduce the clearance of the populationof platelets. As used herein, a population of platelets refers to asample having one or more platelets. A population of platelets includesa platelet concentrate. The term “isolated” means separated from itsnative environment and present in sufficient quantity to permit itsidentification or use. As used herein with respect to a population ofplatelets, isolated means removed or cleared from the blood circulationof a mammal. The circulation time of a population of platelets isdefined as the time when one-half of the platelets in that populationare no longer circulating in a mammal after transplantation into thatmammal. As used herein, “clearance” means removal of the modifiedplatelets from the blood circulation of a mammal (such as but notlimited to by macrophage phagocytosis). As used herein, clearance of apopulation of platelets refers to the removal of a population ofplatelets from a unit volume of blood or serum per unit of time.Reducing the clearance of a population of platelets refers topreventing, delaying, or reducing the clearance of the population ofplatelets. Reducing clearance of platelets also may mean reducing therate of platelet clearance.

A glycan modifying agent refers to an agent that modifies glycanresidues on the platelet. As used herein, a “glycan” or “glycan residue”is a polysaccharide moiety on surface of the platelet, exemplified bythe GP1bα polysaccharide. A “terminal” glycan or glycan residue is theglycan at the distal terminus of the polysaccharide, which typically isattached to polypeptides on the platelet surface. Preferably, the glycanmodifying agent alters GP1bα on the surface of the platelet.

The glycan modifying agents suitable for use as described herein,includes monosaccharides such as arabinose, fructose, fucose, galactose,mannose, ribose, gluconic acid, galactosamine, glucosamine,N-acetylgalactosamine, muramic acid, sialic acid (N-acetylneuraminicacid), and nucleotide sugars such as cytidinemonophospho-N-acetylneuraminic acid (CMP-sialic acid), uridinediphosphate galactose (UDP-galactose) and UDP-galactose precursors suchas UDP-glucose. In some preferred embodiments, the glycan modifyingagent is UDP-galactose or CMP-sialic acid.

UDP-galactose is an intermediate in galactose metabolism, formed by theenzyme UDP-glucose-α-D-galactose-1-phosphate uridylyltransferase whichcatalyzes the release of glucose-1-phosphate from UDP-glucose inexchange for galactose-1-phosphate to make UDP-galactose. UDP-galactoseand sialic acid are widely available from several commercial supplierssuch as Sigma. In addition, methods for synthesis and production ofUDP-galactose are well known in the art and described in the literature(see for example, Liu et al, ChemBioChem 3, 348-355, 2002; Heidlas etal, J. Org. Chem. 57, 152-157; Butler et al, Nat. Biotechnol. 8,281-284, 2000; Koizumi et al, Carbohydr. Res. 316, 179-183, 1999; Endoet al, Appl. Microbiol., Biotechnol. 53, 257-261, 2000). UDP-galactoseprecursors are molecules, compounds, or intermediate compounds that maybe converted (e.g., enzymatically or biochemically) to UDP-galactose.One non-limiting example of a UDP-galactose precursor is UDP-glucose. Incertain embodiments, an enzyme that converts a UDP-galactose precursorto UDP-galactose is added to a reaction mixture (e.g. in a plateletcontainer).

An effective amount of a glycan modifying agent is that amount of theglycan modifying agent that alters a sufficient number of glycanresidues on the surface of platelets, that when introduced to apopulation of platelets, increases circulation time and/or reduces theclearance of the population of platelets in a mammal followingtransplantation of the platelets into the mammal. An effective amount ofa glycan modifying agent is a concentration from about 1 micromolar toabout 1200 micromolar, preferably from about 10 micromolar to about 1000micromolar, more preferably from about 100 micromolar to about 750micromolar, and most preferably from about 200 micromolar to about 600micromolar.

Modification of platelets with glycan modifying agents can be preformedas follows. The population of platelets is incubated with the selectedglycan modifying agent (concentrations of 1-1200 μM) for at least 1, 2,5, 10, 20, 40, 60, 120, 180, 240, or 300 min. at 22° C.-37° C. Multipleglycan modifying agents (i.e., two, three four or more) may be usedsimultaneously or sequentially. In some embodiments 0.1-500 mU/mlgalactose transferase or sialyl transferase is added to the populationof platelets. Galactose transfer can be monitored functionally usingFITC-WGA (wheat germ agglutinin) binding. The goal of the glycanmodification reaction is to reduce WGA binding to resting roomtemperature WGA binding-levels. Galactose transfer can be quantifiedusing ¹⁴C-UDP-galactose. Non-radioactive UDP-galactose is mixed with¹⁴C-UDP-galactose to obtain appropriate galactose transfer. Plateletsare extensively washed, and the incorporated radioactivity measuredusing a γ-counter. The measured cpm permits calculation of theincorporated galactose. Similar techniques are applicable to monitoringsialic acid transfer.

Reducing the clearance of a platelet encompasses reducing clearance ofplatelets after storage at room temperature, or after chilling, as wellas “cold-induced platelet activation”. Cold-induced platelet activationis a term having a particular meaning to one of ordinary skill in theart. Cold-induced platelet activation may manifest by changes inplatelet morphology, some of which are similar to the changes thatresult following platelet activation by, for example, contact withglass. The structural changes indicative of cold-induced plateletactivation are most easily identified using techniques such as light orelectron microscopy. On a molecular level, cold-induced plateletactivation results in actin bundle formation and a subsequent increasein the concentration of intracellular calcium. Actin-bundle formation isdetected using, for example, electron microscopy. An increase inintracellular calcium concentration is determined, for example, byemploying fluorescent intracellular calcium chelators. Many of theabove-described chelators for inhibiting actin filament severing arealso useful for determining the concentration of intracellular calcium(Tsien, R., 1980, supra.). Accordingly, various techniques are availableto determine whether or not platelets have experienced cold-inducedactivation.

The effect of galactose or sialic acid addition to the glycan moietieson platelets, resulting in diminished clearance of modified platelets,can be measured for example using either an in vitro system employingdifferentiated THP-1 cells or murine macrophages, isolated from theperitoneal cavity after thioglycolate injection stimulation. The rate ofclearance of modified platelets compared to unmodified platelets isdetermined. To test clearance rates, the modified platelets are fed tothe macrophages and ingestion of the platelets by the macrophages ismonitored. Reduced ingestion of modified platelets relative tounmodified platelets (twofold or greater) indicates successfulmodification of the glycan moiety for the purposes described herein.

In accordance with the invention, the population of modified plateletscan be chilled without the deleterious effects (cold-induced plateletactivation) usually experienced on chilling of untreated platelets. Thepopulation of modified platelets can be chilled prior to, concurrentlywith, or after contacting the platelets with the at least one glycanmodifying agent. The selective modification of glycan moieties reducesclearance, following chilling (also if not chilled), thus permittinglonger-term storage than is presently possible. As used herein, chillingrefers to lowering the temperature of the population of platelets to atemperature that is less than about 37° C. In some embodiments, theplatelets are chilled to a temperature that is less than about 15° C. Insome preferred embodiments, the platelets are chilled to a temperatureranging from between about 0° C. to about 4° C. Chilling alsoencompasses freezing the platelet preparation, i.e., to temperaturesless than 0° C., −20° C., −50° C., and −80° C. or cooler. Process forthe cryopreservation of cells are well known in the art.

In some embodiments, the population of platelets is stored chilled forat least 3 days. In some embodiments, the population of platelets isstored chilled for at least 5, 7, 10, 14, 21, and 28 days or longer.

In some embodiments of the invention, the circulation time of thepopulation of platelets is increased by at least about 10%. In someother embodiments, the circulation time of the population of plateletsis increased by at least about 25%. In yet some other embodiments, thecirculation time of the population of platelets is increased by at leastabout 50% to about 100%. In still yet other embodiments, the circulationtime of the population of platelets is increased by about 150% orgreater.

The invention also embraces a method for increasing the storage time ofplatelets. As used herein the storage time of platelets is defined asthe time that platelets can be stored without substantial loss ofplatelet function or hemostatic activity such as the loss of the abilityto circulate or increased platelet clearance.

The platelets are collected from peripheral blood by standard techniquesknown to those of ordinary skill in the art, for example by isolationfrom whole blood or by apheresis processes. In some embodiments, theplatelets are contained in a pharmaceutically-acceptable carrier priorto treatment with a glycan modifying agent.

According to another aspect of the invention, a modified platelet or apopulation of modified platelets is provided. The modified plateletcomprises a plurality of modified glycan molecules on the surface of theplatelet. In some embodiments, the modified glycan moieties are GP1bαmolecules. The invention also encompasses a platelet composition in astorage medium. In some embodiments the storage medium comprises apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe platelets and that is a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism.Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art, for example, a buffer that stabilizes the plateletpreparation to a pH of 7.4, the physiological pH of blood, is apharmaceutically acceptable composition suitable for use with thepresent invention.

The invention further embraces a method for making a pharmaceuticalcomposition for administration to a mammal. The method comprisespreparing the above-described platelet preparation, and warming theplatelet preparation. In some embodiments, the method comprisesneutralizing, removing or diluting the glycan modifying agent(s) and/orthe enzyme(s) that catalyze the modification of the glycan moiety, andplacing the modified platelet preparation in a pharmaceuticallyacceptable carrier. In a preferred embodiment, the chilled platelets arewarmed to room temperature (about 22° C.) prior to neutralization ordilution. In some embodiments, the platelets are contained in apharmaceutically acceptable carrier prior to contact with the glycanmodifying agent(s) with or without the enzyme(s) that catalyze themodification of the glycan moiety and it is not necessary to place theplatelet preparation in a pharmaceutically acceptable carrier followingneutralization or dilution.

As used herein, the terms “neutralize” or “neutralization” refer to aprocess by which the glycan modifying agent(s) and/or the enzyme(s) thatcatalyze the modification of the glycan moiety are renderedsubstantially incapable of glycan modification of the glycan residues onthe platelets, or their concentration in the platelet solution islowered to levels that are not harmful to a mammal, for example, lessthat 50 micromolar of the glycan modifying agent. In some embodiments,the chilled platelets are neutralized by dilution, e.g., with asuspension of red blood cells. Alternatively, the treated platelets canbe infused into the recipient, which is equivalent to dilution into ared blood cell suspension. This method of neutralization advantageouslymaintains a closed system and minimizes damage to the platelets. In apreferred embodiment of glycan modifying agents, no neutralization isrequired.

An alternative method to reduce toxicity is by inserting a filter in theinfusion line, the filter containing, e.g. activated charcoal or animmobilized antibody, to remove the glycan modifying agent(s) and/or theenzyme(s) that catalyze the modification of the glycan moiety.

Either or both of the glycan modifying agent(s) and the enzyme(s) thatcatalyze the modification of the glycan moiety also may be removed orsubstantially diluted by washing the modified platelets in accordancewith standard clinical cell washing techniques.

The invention further provides a method for mediating hemostasis in amammal. The method includes administering the above-describedpharmaceutical preparation to the mammal. Administration of the modifiedplatelets may be in accordance with standard methods known in the art.According to one embodiment, a human patient is transfused with redblood cells before, after or during administration of the modifiedplatelets. The red blood cell transfusion serves to dilute theadministered, modified platelets, thereby neutralizing the glycanmodifying agent(s) and the enzyme(s) that catalyze the modification ofthe glycan moiety.

The dosage regimen for mediating hemostasis using the modified plateletsis selected in accordance with a variety of factors, including the type,age, weight, sex and medical condition of the subject, the severity ofthe disease, the route and frequency of administration. An ordinarilyskilled physician or clinician can readily determine and prescribe theeffective amount of modified platelets required to mediate hemostasis.

The dosage regimen can be determined, for example, by following theresponse to the treatment in terms clinical signs and laboratory tests.Examples of such clinical signs and laboratory tests are well known inthe art and are described, see, Harrison's Principles of InternalMedicine, 15th Ed., Fauci A S et al., eds., McGraw-Hill, New York, 2001.

Also within the scope of the invention are storage compositions andpharmaceutical compositions for mediating hemostasis. In one embodiment,the compositions comprise a pharmaceutically-acceptable carrier, aplurality of modified platelets, a plurality of glycan modifyingagent(s) and optionally the enzyme(s) that catalyze the modification ofthe glycan moiety. The glycan modifying agent(s) and the enzyme(s) thatcatalyze the modification of the glycan moiety are present in thecomposition in sufficient amounts so as to reduce platelet clearance.Preferably, glycan modifying agent(s) (and optionally the enzyme(s) thatcatalyze the modification of the glycan moiety) are present in amountswhereby after chilling and neutralization, the platelets maintainsubstantially normal hemostatic activity. The amounts of glycanmodifying agent(s) (and optionally the enzyme(s) that catalyze themodification of the glycan moiety) which reduce platelet clearance canbe selected by exposing a preparation of platelets to increasing amountsof these agents, exposing the treated platelets to a chillingtemperature and determining (e.g., by microscopy) whether or notcold-induced platelet activation has occurred. Preferably, the amountsof glycan modifying agent(s) and the enzyme(s) that catalyze themodification of the glycan moiety can be determined functionally byexposing the platelets to varying amounts of glycan modifying agent(s)and the enzyme(s) that catalyze the modification of the glycan moiety,chilling the platelets as described herein, warming the treated(chilled) platelets, optionally neutralizing the platelets and testingthe platelets in a hemostatic activity assay to determine whether thetreated platelets have maintained substantially normal hemostaticactivity.

For example, to determine the optimal concentrations and conditions forpreventing cold-induced activation of platelets by modifying them with aglycan modifying agent(s) (and optionally the enzyme(s) that catalyzethe modification of the glycan moiety), increasing amounts of theseagents are contacted with the platelets prior to exposing the plateletsto a chilling temperature. The optimal concentrations of the glycanmodifying agent(s) and the enzyme(s) that catalyze the modification ofthe glycan moiety are the minimal effective concentrations that preserveintact platelet function as determined by in vitro tests (e.g.,observing morphological changes in response to glass, thrombin,cryopreservation temperatures; ADP-induced aggregation) followed by invivo tests indicative of hemostatic function (e.g., recovery, survivaland shortening of bleeding time in a thrombocytopenic animal or recoveryand survival of ⁵¹Cr-labeled platelets in human subjects).

According to yet another aspect of the invention, a composition foraddition to platelets to reduce platelet clearance or to increaseplatelet storage time is provided. The composition includes one or moreglycan modifying agents. In certain embodiments, the composition alsoincludes an enzyme(s) that catalyze the modification of the glycanmoiety. The glycan modifying agent and the enzyme(s) that catalyzes themodification of the glycan moiety are present in the composition inamounts that prevent cold-induced platelet activation.

The invention also embraces a storage composition for preservingplatelets. The storage composition comprises at least one glycanmodifying agent in an amount sufficient to reduce platelet clearance. Insome embodiments the storage composition further comprises an enzymethat catalyzes the modification of a glycan moiety on the platelet. Theglycan modifying agent is added to the population of platelets that arepreferably kept between about room temperature and 37° C. In someembodiments, following treatment, the population of platelets is cooledto about 4° C. In some embodiments, the platelets are collected into aplatelet pack, bag, or container according to standard methods known toone of skill in the art. Typically, blood from a donor is drawn into aprimary container which may be joined to at least one satellitecontainer, all of which containers are connected and sterilized beforeuse. In some embodiments, the satellite container is connected to thecontainer for collecting platelets by a breakable seal. In someembodiments, the primary container further comprises plasma containing aplurality of platelets.

In some embodiments, the platelets are concentrated (e.g. bycentrifugation) and the plasma and red blood cells are drawn off intoseparate satellite bags (to avoid modification of these clinicallyvaluable fractions) prior to adding the glycan modifying agent with orwithout the enzyme that catalyzes the modification of a glycan moiety onthe platelet. Platelet concentration prior to treatment also mayminimize the amounts of glycan modifying agents required for reducingthe platelet clearance, thereby minimizing the amounts of these agentsthat are eventually infused into the patient.

In one embodiment, the glycan modifying agent(s) are contacted with theplatelets in a closed system, e.g. a sterile, sealed platelet pack, soas to avoid microbial contamination. Typically, a venipuncture conduitis the only opening in the pack during platelet procurement ortransfusion. Accordingly, to maintain a closed system during treatmentof the platelets with the glycan modifying agent(s), the agent(s) isplaced in a relatively small, sterile container which is attached to theplatelet pack by a sterile connection tube (see e.g., U.S. Pat. No.4,412,835, the contents of which are incorporated herein by reference).The connection tube may be reversibly to sealed, or have a breakableseal, as will be known to those of skill in the art. After the plateletsare concentrated, e.g. by allowing the platelets to settle and squeezingthe plasma out of the primary pack and into a second bag according tostandard practice, the seal to the container(s) including the glycanmodifying agent(s) is opened and the agents are introduced into theplatelet pack. In one embodiment, the glycan modifying agents arecontained in separate containers having separate resealable connectiontubes to permit the sequential addition of the glycan modifying agentsto the platelet concentrate.

Following contact with the glycan modifying agent(s), the treatedplatelets are chilled. In contrast to platelets stored at, for example,22° C., platelets stored at cryopreservation temperatures havesubstantially reduced metabolic activity. Thus, platelets stored at 4°C. are metabolically less active and therefore do not generate largeamounts of CO₂ compared with platelets stored at, for example, 22° C.(Slichter, S. J., 1981, Vox Sang 40 (Suppl 1), pp 72-86, ClinicalTesting and Laboratory-Clinical correlations.). Dissolution of CO₂ inthe platelet matrix results in a reduction in pH and a concomitantreduction in platelet viability (Slichter, S., 1981, supra.). This canbe resolved by adding buffers to the platelet population, the buffersselected to keep the platelet population at or near the physiological pHof blood. Likewise, conventional platelet packs are formed of materialsthat are designed and constructed of a sufficiently permeable materialto maximize gas transport into and out of the pack (O₂ in and CO₂ out).The prior art limitations in platelet pack design and construction areobviated by the instant invention, which permits storage of platelets atcryopreservation temperatures, thereby substantially reducing plateletmetabolism and diminishing the amount of CO₂ generated by the plateletsduring storage. Accordingly, the invention further provides plateletcontainers that are substantially non-permeable to CO₂ and/or O₂, whichcontainers are useful particularly for cold storage of platelets. Inboth the gas permeable and non-gas permeable embodiments, the inventionprovides for a blood storage container having therein, a quantity of aglycan modifying agent sufficient to substantially modify thecarbohydrates of the platelets introduced therein, such that theplatelets become capable of cold storage and subsequent in vivocirculation.

The present invention also provides for kits that are used for plateletcollection, processing and storage, further including suitable packagingmaterials and instructions for using the kit contents. It is preferredthat all reagents and supplies in the kit be sterile, in accordance withstandard medical practices involving the handling and storage of bloodand blood products. Methods for sterilizing the kit contents are knownin the art, for example, ethylene gas, irradiation and the like. Incertain embodiments, the kit may include venipuncture supplies and/orblood collection supplies, for example a needle set, solution forsterilizing the skin of a platelet donor, and a blood collection bag orcontainer. Preferably the container is “closed”, i.e., substantiallysealed from the environment. Such closed blood collection containers arewell known in the art, and provide a means of preventing microbialcontamination of the platelet preparation contained therein. Otherembodiments include kits containing supplies for blood collection andplatelet apheresis. The kits may further include a quantity of theglycan modifying agent, sufficient to modify the volume of plateletscollected and stored in the container. In certain embodiments, the kitincludes reagents for modifying the terminal glycan of platelets with asecond or third chemical moiety, for example to PEGylate collectedplatelets. In other embodiments, the kit includes a blood collectionsystem having a blood storage container wherein the glycan modifyingagent is provided within the container in an amount sufficient to treatthe volume of blood or platelets held by the container. The quantity ofglycan modifying agent will depend on the volume of the container. It ispreferred the glycan modifying agent be provided as a sterilenon-pyogenic solution, but it may also be supplied as a lyophilizedpowder. For example, a blood bag is provided having a capacity of 250ml. Contained in the blood bag is a quantity of UDP-Gal such that when250 ml of blood is added, the final concentration of the UDP-Gal isapproximately 200 micromolar. Other embodiments contain differentconcentrations of glycan modifying agents, for example but not limitedto quantities resulting in final concentrations of 10 micromolar to 10millimolar, and preferably 100 micromolar to 1 millimolar of the glycanmodifying agents. Other embodiments use combinations of glycan modifyingagents, e.g., to effect sialyation or galactosylation of N-linkedglycoproteins on blood products introduced into the container.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Introduction

Modest cooling primes platelets for activation, but refrigeration causesshape changes and rapid clearance, compromising storage of platelets fortherapeutic transfusions. We found that shape change inhibition does notnormalize cold-induced clearance. We also found that cooling plateletsrearranges the surface configuration of the von Willebrand factor (vWf)receptor complex α subunit (GP1bα) such that it becomes targeted forrecognition by complement receptor 3 receptors (CR3) predominantlyexpressed on liver macrophages, leading to platelet phagocytosis andclearance. GP1bα removal prolongs survival of unchilled platelets.Chilled platelets bind vWf and function normally in vitro and ex vivoafter transfusion into CR3-deficient mice. Cooled platelets, however,are not “activated” like platelets exposed to thrombin or ADP, and theirvWf-receptor complex reacts normally with activated vWf.

As the temperature falls below 37° C. platelets become more susceptibleto activation by thrombotic stimuli, a phenomenon known as “priming”(Faraday and Rosenfeld, 1998; Hoffmeister et al., 2001). Priming may bean adaptation to limit bleeding at lower temperatures of body surfaceswhere most injuries occur. We propose that the hepatic clearancesystem's purpose is to remove repeatedly primed platelets, and thatconformational changes in GP1bα that promote this clearance do notaffect GP1bα's hemostatically important binding to vWf. Therefore,selective modification of GP1bα may accommodate cold storage ofplatelets for transfusion.

Materials and Methods

We obtained fluorescein isothiocyanate (FITC)-conjugated annexin V,phycoerythrin (PE)-conjugated anti-human CD11b/Mac-1 monoclonalantibodies (mAb), FITC-conjugated anti-mouse and anti-human IgM mAb,FITC-conjugated anti-mouse and anti-human CD62P-FITC mAb from Pharmingen(San Diego, Calif.); FITC-conjugated rat anti-mouse anti-human IgG mAbfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.);FITC-conjugated anti-human CD61 mAbs (clone BL-E6) from AccurateScientific Corp. (Westbury, N.Y.); FITC-conjugated anti-human GP1bα mAb(clone SZ2) from Immunotech (Marseille, France); and FITC-conjugatedpolyclonal rabbit anti-vWf antibody from DAKOCytomation (Glostrup,Denmark). We purchased EGTA-acetoxymethylester (AM), Oregon Greencoupled fibrinogen from human plasma, CellTracker™ Orange CMTMR;CellTracker Green CMFDA, Nile-red (535/575) coupled andcarboxylate-modified 1 μm microspheres/FluoSpheres from MolecularProbes, Inc. (Eugene, Oreg.) and ¹¹¹Indium from NEN Life ScienceProducts (Boston, Mass.). We purchased Cytochalasin B, dimethylsulfoxide (DMSO), trisodium isothiocyanate (TRITC), human thrombin,prostaglandin E1 (PGE₁), phorbol ester 12-tetradecanoylphorbol-13acetate (PMA), A23187 ionophore from Sigma (St. Louis, Mo.); botrocetinfrom Centerchem Inc. (Norwalk, Conn.); andO-sialoglycoprotein-endopeptidase from Cerladane (Hornby, Canada). HBSScontaining Ca²⁺ and Mg²⁺, pH 6.4; RPMI 1640; 0.05% Trypsin-EDTA (0.53mM) in HBSS without Ca²⁺ and Mg²⁺; and other supplements (penicillin,streptomycin and fetal bovine serum) were from GIBCO Invitrogen Corp.(Grand Island, N.Y.). TGF-β1 from Oncogene to Research Products(Cambridge, Mass.); 1,25-(OH)₂ vitamin D3 from Calbiochem (San Diego,Calif.); and Adenosine-5′-Diphosphate (ADP) were from USB (Cleveland,Ohio). Avertin (2,2,2-tribromoethanol) was purchased from Fluka Chemie(Steinheim, Germany). Collagen related peptide (CRP) was synthesized atthe Tufts Core Facility, Physiology Dept. (Boston, Mass.) andcross-linked as previously described (Morton et al., 1995). Mocarhagin,a snake venom metalloprotease, was provided by Dr. M. Berndt, BakerMedical Research Institute, Melbourne Victoria 318 1, Australia.Additional unconjugated anti mouse GP1bα mAbs and a PE-conjugatedanti-mouse GP1bα mAb pOp4 were provided by Dr. B. Nieswandt(Witten/Herdecke University, Wuppertal, Germany). We obtained THP-1cells from the American Type Culture Collection (Manassas, Va.).

Animals

For assays of clearance and survival studies, we used age-, strain- andsex-matched C57BL/6 and C57BL/6×129/sv wild type mice obtained fromJackson Laboratory (Bar Harbor, Me.). C57BL/6×129/sv mice deficient incomplement component C3 (Wessels et al., 1995) were provided by Dr. M.C. Carroll (Center for Blood Research and Department of Pediatrics,Harvard Medical School, Boston, Mass.). C57BL/6 mice deficient in CR3(Coxon et al., 1996) were provided by Dr. T Mayadas and C57BL/6 micedeficient in vWf (Denis et al., 1998) were provided by Dr. D. Wagner.Mice were maintained and treated as approved by Harvard Medical AreaStanding Committee on Animals according to NIH standards as set forth inThe Guide for the Care and Use of Laboratory Animals.

Human Platelets

Blood was drawn from consenting normal human volunteers (approval wasobtained from the Institutional Review Boards of both Brigham andWomen's Hospital and the Center for Blood Research (Harvard MedicalSchool)) by venipuncture into 0.1 volume of Aster-Jandl citrate-basedanticoagulant (Hartwig and DeSisto, 1991) and platelet rich plasma (PRP)was prepared by centrifugation of the anticoagulated blood at 300×g for20 min at room temperature. Platelets were separated from plasmaproteins by gel-filtration at room temperature through a small Sepharose2B column (Hoffmeister et al., 2001). Platelets used in the in vitrophagocytosis assay described below were labeled with 1.8 μM CellTracker™Orange CMTMR (CM-Orange) for 20 min at 37° C. (Brown et al., 2000), andunincorporated dye was removed by centrifugation (850×g, 5 min.) with 5volumes of washing buffer containing 140 mM NaCl, 5 mM KCl, 12 mMtrisodium citrate, 10 mM glucose, and 12.5 mM sucrose, 1 μg/ml PGE₁, pH6.0 (buffer A). Platelets were resuspended at 3×10⁸/ml in a solutioncontaining 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mMglucose and 10 mM Hepes, pH 7.4 (buffer B).

The N-terminus of GP1bα was enzymatically removed from the surface ofchilled or room temperature maintained and labeled platelets in bufferB, also containing 1 mM Ca²⁺ and 10 μg/ml of the snake venommetalloprotease mocarhagin (Ward et al., 1996). After the enzymaticdigestion, the platelets were washed by centrifugation with 5× volume ofbuffer A and routinely checked by microscopy for aggregates.GP1bα-N-terminus removal was monitored by incubating plateletsuspensions with 5 μg/ml of FITC-conjugated anti-human GP1bα (SZ2) mAbfor 10 min at room temperature and followed by immediate flow cytometryanalysis on a FACScalibur Flow Cytometer (Becton Dickinson Biosciences,San Jose, Calif.). Platelets were gated by forward/side scattercharacteristics and 50,000 events acquired.

Murine Platelets

Mice were anesthetized with 3.75 mg/g (2.5%) of Avertin, and 1 ml bloodwas obtained from the retroorbital eye plexus into 0.1 volume ofAster-Jandl anticoagulant. PRP was prepared by centrifugation ofanticoagulated blood at 300×g for 8 min at room temperature. Plateletswere separated from plasma proteins by centrifugation at 1200×g for 5min and washed two times by centrifugation (1200×g for 5 min) using 5×volumes of washing buffer (buffer A). This procedure is meant bysubsequent use of the term “washed”. Platelets were resuspended at aconcentration of 1×10⁹/ml in a solution containing 140 mM NaCl, 3 mMKCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose and 10 mM Hepes, pH 7.4(buffer B). Platelet count was determined using a Bright LineHemocytometer (Hausser Scientific, Horsham, Pa.) under a phase-contrastmicroscope at 400× magnification. Some radioactive platelet clearancestudies were performed with ¹¹¹Indium, and we labeled mouse plateletsusing a method described for primate platelets (Kotze et al., 1985).Platelets were resuspended at a concentration of 2×10⁹/ml in 0.9% NaCl,pH 6.5 (adjusted with 0.1 M sodium citrate), followed by the addition of500 μCi ¹¹¹Indium chloride for 30 min at 37° C. and washed as describedabove and suspended in buffer B at a concentration of 1×10⁹/ml.

For intravital microscopy or other platelet survival experiments, washedplatelets were labeled either with 2.5 μM CellTracker Green CMFDA(5-chloromethyl fluorescein diacetate) (CMFDA) for 20 min at 37° C.(Baker et al., 1997) or with 0.15 μM TRITC for 20 min at 37° C. inbuffer B also containing 0.001% DMSO, 20 mM HEPES. Unincorporated dyewas removed by centrifugation as described above, and platelets weresuspended at a concentration of 1×10⁹/ml in buffer B.

The N-terminus of GP1bα was enzymatically removed from the surface ofchilled or room temperature labeled platelets with 100 μg/mlO-sialoglycoprotein endopeptidase in buffer B containing 1 mM Ca²⁺ for20 min at 37° C. (Bergmeier et al., 2001). After enzymatic digestion,platelets were washed by centrifugation and checked by light microscopyfor aggregates. Enzymatic removal of the GP1bα-N-terminus removal wasmonitored by incubating the platelet suspensions with 5 μg/ml ofPE-conjugated anti-mouse GP1bα mAb pOp4 for 10 min at room temperature,and bound PE analyzed by flow cytometry.

To inhibit cold-induced platelet shape changes, 10⁹/ml platelets inbuffer B were loaded with 2 μM EGTA-AM followed by 2 μM cytochalasin Bas previously described (Winokur and Hartwig, 1995), labeled with 2.5 μMCMFDA for 30 min at 37° C. and then chilled or maintained at roomtemperature. The platelets were subjected to standard washing andsuspended at a concentration of 1×10⁹/ml in buffer B before injectioninto mice.

Platelet Temperature Protocols

To study the effects of temperature on platelet survival or function,unlabeled, radioactively labeled, or fluorescently-labeled mouse orhuman platelets were incubated for 2 hours at room temperature (25-27°C.) or else at ice bath temperatures and then rewarmed for 15 minutes at37° C. before transfusion into mice or in vitro analysis. Plateletssubjected to these treatments are designated cooled or chilled (orchilled, rewarmed) and room temperature platelets respectively.

Murine Platelet Recovery, Survival and Fate

CMFDA labeled chilled or room temperature murine platelets (10⁸) wereinjected into syngeneic mice via the lateral tail vein using a 27-gaugeneedle. For recovery and survival determination, blood samples werecollected immediately (<2 min) and 0.5, 2, 24, 48, 72 hours aftertransfusion into 0.1 volume of Aster-Jandl anticoagulant. Whole bloodanalysis using flow cytometry was performed and the percentage of CMFDApositive platelets determined by gating on all platelets according totheir forward and side scatter characteristics (Baker et al., 1997).50,000 events were collected in each sample. CMFDA positive plateletsmeasured at a time <2 min was set as 100%. The input of transfusedplatelets per mouse was ˜2.5-3% of the whole platelet population.

To evaluate the fate of platelets, tissues (heart, lung, liver, spleen,muscle, and femur) were harvested at 0.5, 1 and 24 hours after theinjection of 10⁸ chilled or room temperature ¹¹¹Indium labeled plateletsinto mice. The organ-weight and their radioactivity were determinedusing a Wallac 1470 Wizard automatic gamma counter (Wallac Inc.,Gaithersburg, Md.). The data were expressed as gamma count per gramorgan. For recovery and survival determination of radioactive platelets,blood samples were collected immediately (<2 min) and 0.5 and hoursafter transfusion into 0.1 volume of Aster-Jandl anticoagulant and theirgamma counts determined (Kotze et al., 1985).

Platelet Aggregation

Conventional tests were performed and monitored in a Bio/Dataaggregometer (Horsham, Pa.). Samples of 0.3-ml murine washed and stirredplatelets were exposed to 1 U/ml thrombin, 10 μM ADP, or 3 μg/ml CRP at37° C. Light transmission was recorded over 3 min.

Activated VWf Binding

Platelet rich plasma was treated with or without 2 U/ml botrocetin for 5min at 37° C. (Bergmeier et al., 2001). Bound vWf was detected by flowcytometry using FITC conjugated polyclonal rabbit anti-vWf antibody.

Surface labeling of platelet GP1bα

Resting mouse platelets maintained at room temperature or chilled 2 hrswere diluted to a concentration of 2×10⁶/ml in phosphate buffered saline(PBS) containing 0.05% glutaraldehyde. Platelet solutions (200 μl) wereplaced on a polylysine-coated glass coverslip contained in wells of96-well plate, and the platelets were adhered to each coverslip bycentrifugation at 1,500×. g for 5 min at room temperature. Thesupernatant fluid was then removed, and platelets bound to the coverslipwere fixed with 0.5% glutaraldehyde in PBS for 10 min. The fixative wasremoved, unreacted aldehydes quenched with a solution containing 0.1%sodium borohydride in PBS followed by washing with PBS containing 10%BSA. GP1bα on the platelet surface was labeled with a mixture of threerat anti-mouse GP1bα monoclonal antibodies, each at 10 μg/ml (Bergmeieret al., 2000) for 1 hr followed by 10 nm gold coated with goat anti-ratIgG. The coverslips were extensively washed with PBS, post-fixed with 1%glutaraldehyde, washed again with distilled water, rapidly frozen,freeze-dried, and rotary coated with 1.2 nm of platinum followed by 4 nmof carbon without rotation in a Cressington CFE-60 (Cressington,Watford, UK). Platelets were viewed at 100 kV in a JEOL 1200-EX electronmicroscope (Hartwig et al., 1996; Kovacsovics and Hartwig, 1996)

In Vitro Phagocytic Assay

Monocytic THP-1 cells were cultured for 7 days in RPMI 1640 cell culturemedia supplemented with 10% fetal bovine serum, 25 mM Hepes, 2 mMglutamine and differentiated using 1 ng/ml TGFβ and 50 nM 1,25-(OH)₂vitamin D3 for 24 hours, which is accompanied by increased expression ofCR3 (Simon et al., 2000). CR3 expression was monitored by flow cytometryusing a PE-conjugated anti-human CD11b/Mac-1 mAb. Undifferentiated ordifferentiated THP-1 cells (2×10⁶/ml) were plated onto 24-well platesand allowed to adhere for 45 minutes at 37° C. The adherentundifferentiated or differentiated macrophages were activated by theaddition of 15 ng/ml PMA for 15 min. CM-range-labeled, chilled or roomtemperature platelets (10⁷/well), previously subjected to differenttreatments were added to the undifferentiated or differentiatedphagocytes in Ca²⁺- and Mg²⁺-containing HBSS and incubated for 30 min at37° C. Following the incubation period, the phagocyte monolayer waswashed with HBSS for 3 times, and adherent platelets were removed bytreatment with 0.05% trypsin/0.53 mM EDTA in HBSS at 37° C. for 5 minfollowed by 5 mM EDTA at 4° C. to detach the macrophages for flowcytometric analysis of adhesion or ingestion of platelets (Brown et al.,2000). Human CM-Orange-labeled, chilled or room temperature plateletsall expressed the same amount of the platelet specific marker CD61 asfreshly isolated unlabeled platelets (not shown). CM-Orange-labeledplatelets incubated with macrophages were resolved from the phagocytesaccording to their forward and side scatter properties. The macrophageswere gated, 10,000 events acquired for each sample, and data analyzedwith CELLQuest software (Becton Dickenson). CM-Orange-labeled plateletsthat associate with the phagocyte population have a shift in orangefluorescence (FIG. 6 a and FIG. 6 b, ingested, y axis). These plateletswere ingested rather than merely adherent, because they failed to duallabel with the FITC-conjugated mAb to CD61.

Immunolabeling and Flow Cytometry of Platelets

Washed murine or human platelets (2×10⁶) were analyzed for surfaceexpression of CD62P, CD61, or surface bound IgM and IgG after chillingor room temperature storage by staining with fluorophore-conjugated Abs(5 μg/ml) for 10 min at 37° C. Phosphatidylserine exposure by chilled orroom temperature platelets was determined by resuspending 5 μl ofplatelets in 400 μl of HBSS containing 10 mM Ca²⁺ with 10 μg/ml ofFITC-conjugated annexin-V. As a positive control for PS exposure,platelet suspensions were stimulated with 1 μM A23187. Fibrinogenbinding was determined by the addition of Oregon Green-fibrinogen for 20min at room temperature. All platelet samples were analyzed immediatelyby flow cytometry. Platelets were gated by forward and side scattercharacteristics.

Intravital Microscopy Experiments

Animal preparation, technical and experimental aspects of the intravitalvideo microscopy setup have been described (von Andrian, 1996). Six toeight week-old mice of both sexes were anesthetized by intraperitonealinjection of a mixture of Xylazine and Ketamin. The right jugular veinwas catheterized with PE-10 polyethylene tubing. The lower surface ofthe left liver lobe was surgically prepared and covered by a glass coverslip for further in vivo microscopy as described (McCuskey, 1986). 10⁸chilled platelets and room temperature platelets labeled with CMFDA andTRITC respectively were mixed 1:1 and administered intravenously. Thecirculation of labeled platelets in liver sinusoids was followed byvideo triggered stroboscopic epi-illumination. Ten video scenes wererecorded from 3 centrilobular zones at each indicated time point. Theratio of cooled (CMFDA)/RT (TRITC) adherent platelets in the identicalvisualized field was calculated. Confocal microscopy was performed usinga Radiance 2000 MP confocal-multiphoton imaging system connected to anOlympus BX 50 WJ upright microscope (Biorad, Hercules, Calif.), using a10× water immersion objective. Images were captured and analyzed withLaser Sharp 2000 software (Biorad) (von Andrian, 2002).

Platelet Aggregation in Shed Blood

We used a flow cytometric method to analyze aggregate formation byplatelets in whole blood emerging from a wound as described for primates(Michelson et al., 1994). We injected 10⁸ CMFDA labeled room temperaturemurine platelets into syngeneic wild type mice and 10⁸ CMFDA labeled,chilled platelets into CR3-deficient mice. Twenty-four hours after theplatelet infusion, a standard bleeding time assay was performed,severing a 3-mm segment of a mouse tail (Denis et al., 1998). Theamputated tail was immersed in 100 μl 0.9% isotonic saline at 37° C. Theemerging blood was collected for 2 min., and 0.1 volume of Aster-Jandlanticoagulant added and followed immediately with 1% paraformaldehyde(final concentration). Peripheral blood was obtained by retroorbital eyeplexus bleeding in parallel as described above and immediately fixedwith 1% paraformaldehyde (final concentration). To analyze the number ofaggregates in vive by flow cytometry, the shed blood emerging from thebleeding time wound, as well as a peripheral whole blood sample, werediluted and labeled with PE-conjugated anti-murine GP1bα mAb pOp4 (5μg/ml, 10 min.). Platelets were discriminated from red cells and whitecells by gating according to their forward scatter characteristics andGP1bα positivity. A histogram of log forward light scatter (reflectingplatelet size) versus GP1bα binding was then generated. In theperipheral whole blood samples, analysis regions were plotted around theGP1bα-positive particles to include 95% of the population on the forwardscatter axis (region 1) and the 5% of particles appearing above thisforward light scatter threshold (region 2). Identical regions were usedfor the shed blood samples. The number of platelet aggregates in shedblood as a percentage of the number of single platelets was calculatedfrom the following formula: [(number of particles in region 2 of shedblood)−(number of particles in region 2 of peripheral blood)]÷(number ofparticles in region 1 of shed blood)×100%. The infused platelets wereidentified by their CMFDA labeling and discriminated from the CMFDAnegative non-infused platelets.

Flow Cytometric Analysis of Murine Platelet Fibrinogen Binding andP-Selectin Exposure of Circulating Platelets

Room temperature CM-Orange-labeled room temperature platelets (10⁸) wereinjected into wild type mice and CM-Orange-chilled labeled platelets(10⁸) into CR3 deficient mice. Twenty-four hours after platelet infusionthe mice were bled and the platelets isolated. Resting or thrombinactivated (1 U/ml, 5 min) platelet suspensions (2×10⁸) were diluted inPBS and either stained with FITC-conjugated anti-mouse P-selectin mAb orwith 50 μg/ml Oregon Green-conjugated fibrinogen for 20 min at roomtemperature. Platelet samples were analyzed immediately by flowcytometry. Transfused and non-transfused platelets were gated by theirforward scatter and CM-Orange fluorescence characteristics. P-selectinexpression and fibrinogen binding were measured for each CM-Orangepositive and negative population before and after stimulation withthrombin.

Statistics

The intravital microscopy data are expressed as means±SEM. Groups werecompared using the nonpaired t test. P values<0.05 were consideredsignificant. All other data are presented as the mean±SD.

Results The Clearance of Chilled Platelets Occurs Predominantly in theLiver and is Independent of Platelet Shape.

Mouse platelets kept at room temperature (RT) and infused into syngeneicmice disappear at fairly constant rate over time for about 80 hours(FIG. 1A). In contrast, approximately two-thirds of mouse plateletschilled at ice-bath temperature and rewarmed (Cold) before injectionrapidly disappear from the circulation as observed previously in humansand mice (Becker et al., 1973; Berger et al., 1998). Chilled andrewarmed platelets treated with the cell-permeable calcium chelatorEGTA-AM and the actin filament barbed end capping agent cytochalasin B(Cold+CytoB/EGTA) to preserve their discoid shape (Winokur and Hartwig,1995), left the circulation as rapidly as chilled, untreated plateletsdespite the fact that these platelets were fully functional asdetermined by thrombin-, ADP- or collagen related peptide-(CRP) inducedaggregation in vitro (FIG. 1B). The recoveries of infused plateletsimmediately following transfusion were 50-70%, and the kinetics ofplatelet disappearance were indistinguishable whether we used ¹¹¹Indiumor CMFDA to label platelets. The relative survival rates of roomtemperature and chilled mouse platelets resemble the values reportedpreviously for identically treated mouse (Berger et al., 1998) and humanplatelets (Becker et al., 1973).

FIG. 1C shows that the organ destinations of room temperature andchilled mouse platelets differ. Whereas room-temperature plateletsprimarily end up in the spleen, the liver is the major residence ofchilled platelets removed from the circulation. A greater fraction ofradionuclide detected in the kidneys of animals receiving¹¹¹Indium-labeled chilled compared with room-temperature platelets at 24hours may reflect a more rapid degradation of chilled platelets anddelivery of free radionuclide to the urinary system. One hour afterinjection the organ distribution of platelets labeled with CMFDA wascomparable to that of platelets labeled with ¹¹¹Indium. In both cases,60-90% of the labeled chilled platelet population deposited in theliver, ˜20% in the spleen and ˜15% in the lung. In contrast, a quarterof the infused room temperature platelets distributed equally among theliver, spleen and lung.

Chilled Platelets Co-Localize with Liver Macrophages (Kupffer Cells).

The clearance of chilled platelets by the liver and the evidence forplatelet degradation is consistent with recognition and ingestion ofchilled platelets by Kupffer cells, the major phagocytic scavenger cellsof the liver. FIG. 1D shows the location of phagocytotic Kupffer cellsand adherent chilled CMFDA-labeled platelets in a representativeconfocal micrograph of a mouse liver section 1 hour after transfusion.Sinusoidal macrophages were visualized by the injection of 1 μm carboxylmodified polystyrene microspheres marked with Nile-red. Co-localizationof transfused platelets and macrophages is indicated in yellow in themerged micrograph of both fluorescence emissions. The chilled plateletslocalize with Nile-red-labeled cells preferentially in the periportaland midzonal domains of liver acini, sites rich in sinusoidalmacrophages (Bioulac-Sage et al., 1996; MacPhee et al., 1992).

CR3-Deficient Mice do not Rapidly Clear Chilled Platelets.

CR3 (α_(M)β₂ integrin; CD11b/CD18; Mac-1) is a major mediator ofantibody independent clearance by hepatic macrophages. FIG. 2 a showsthat chilled platelets circulate in CR3-deficient animals with the samekinetics as room-temperature platelets, although the clearance of bothplatelet populations is shorter in the CR3-deficient mouse compared tothat in wild-type mice (FIG. 1 a). The reason for the slightly fasterplatelet removal rate by CR3-deficient mice compared to wild-type miceis unclear. Chilled and rewarmed platelets also clear rapidly fromcomplement factor 3 C3-deficient mice (FIG. 2 c), missing a majoropsonin that promotes phagocytosis and clearance via CR3 and from vonWillebrand factor (vWf) deficient mice (Denis et al., 1998) (FIG. 2 b).

Chilled Platelets Adhere Tightly to Kupffer Cells In Vivo.

Platelet adhesion to wild-type liver sinusoids was further investigatedby intravital microscopy, and the ratio between chilled and roomtemperature stored adherent platelets infused together was determined.FIG. 3A-3C show that both chilled and room temperature platelets attachto sinusoidal regions with high Kupffer cell density (FIGS. 3 a and 3b), but that 2.5 to 4-times more chilled platelets attach to Kupffercells in the wild-type mouse than room-temperature platelets (FIG. 3 c).In contrast, the number of platelets adhering to Kupffer cells inCR3-deficient mice was independent of chilling or room temperatureexposure (FIG. 3 c).

Chilled Platelets Lacking the N-Terminal Domain of GP1bα CirculateNormally.

Because GP1bα, a component of the GP1b-IX-V receptor complex for vWf,can bind CR3 under certain conditions in vitro (Simon et al., 2000), weinvestigated GP1bα as a possible counter receptor on chilled plateletsfor CR3. The 0-sialoglycoprotein endopeptidase cleaves the 45-kDaN-terminal extracellular domain of the murine platelet GP1bα, leavingother platelet receptors such as (α_(IIb)β₃, α₂α₁, GPVI/FcRγ-chain andthe protease-activated receptors intact (Bergmeier et al., 2001). Hence,we stripped this portion of the extracellular domain of GP1bα from mouseplatelets with 0-sialoglycoprotein endopeptidase (FIG. 4A inset) andexamined their survival in mice following room temperature or coldincubation. FIG. 4A shows that chilled platelets no longer exhibit rapidclearance after cleavage of GP1bα. In addition, GP1bα depleted roomtemperature-treated platelets have slightly elongated survival times(˜5-10%) when compared to the GP1bα-containing room-temperaturecontrols.

Chilling does not Affect Binding of Activated vWf to the PlateletvWf-Receptor but Induces Clustering of GP1bα on the Platelet Surface.

FIG. 4B shows that botrocetin-activated vWf binds GP1bα equally well onroom temperature as on cold platelets, although chilling of plateletsleads to changes in the distribution of GP1bα on the murine plateletsurface. GP1bα molecules, identified by immunogold labeled monoclonalmurine anti-GP1bα antibodies, form linear aggregates on the smoothsurface of resting discoid platelets at room temperature (FIG. 4C, RT).This arrangement is consistent with information about the architectureof the resting blood platelet. The cytoplasmic domain of GP1bα bindslong filaments curving with the plane of the platelet membrane throughthe intermediacy of filamin A molecules (Hartwig and DeSisto, 1991).After chilling (FIG. 4C, Chilled) many GP1bα molecules organize asclusters over the platelet membrane deformed by internal actinrearrangements (Hoffmeister et al., 2001; Winokur and Hartwig, 1995).

Recognition of Platelet GP1bα by CR3-Mediates Phagocytosis of ChilledHuman Platelets In Vitro.

Differentiation of human monocytoid THP-1 cells using TGF-β1 and1,25-(OH)₂ Vitamin D3 increases expression of CR3 by ˜2-fold (Simon etal., 1996). Chilling resulted in 3-fold increase of plateletphagocytosis by undifferentiated THP-1 cells and a −5-fold increase bydifferentiated THP-1 cells (FIGS. 5B and 5 c), consistent with mediationof platelet uptake by CR3. In contrast, the differentiation of THP-1cells had no significant effect on the uptake of room temperature storedplatelets (FIGS. 5A and 5 c). To determine if GP1bα is the counterreceptor for CR3-mediated phagocytosis on chilled human platelets, weused the snake venom metalloprotease mocarhagin, to remove theextracellular domain of GP1bα (Ward et al., 1996). Removal of humanGP1bα from the surface of human platelets with mocarhagin reduced theirphagocytosis after chilling by ˜98% (FIG. 5C).

Exclusion of Other Mediators of Cold-Induced Platelet Clearance

Table 1 shows results of experiments that examined whether coolingaffected the expression of platelet receptors other than GP1bα or theirinteraction with ligands. These experiments revealed no detectableeffects on the expression of P-selectin, α_(IIb)β₃-integrin density oron α_(IIb)β₃ fibrinogen binding, a marker of α_(IIb)β₃ activation.Chilling also did not increase phosphatidylserine (PS) exposure, anindicator of apoptosis, nor did it change platelet binding of IgG or IgMimmunoglobulins.

TABLE 1 Effect of chilling on binding of various antibodies or ligandsto platelet receptors. Binding ratio 4° C.:22° C. Platelet receptor(ligand) Human platelets Murine platelets P-Selectin (anti-CD62P mAb)1.01 ± 0.06 1.02 ± 0.03 Platelet associated IgGs 1.05 ± 0.14 1.06 ± 0.03Platelet associated IgMS 0.93 ± 0.10 1.01 ± 0.02 Phosphatidylserine(annexin V) 0.95 ± 0.09 1.04 ± 0.02 α_(IIb)β₃ anti-CD61 mAb) 1.03 ± 0.051.04 ± 0.10 α_(IIb)β₃ (fibrinogen) 1.05 ± 0.10 1.06 ± 0.06

The binding of fluorescently labeled antibodies or ligands againstvarious receptors on chilled-rewarmed or room temperature human andmurine platelets was measured by flow cytometry. The data are expressedas the ratio between the mean fluorophore bound to the surface ofchilled versus room temperature platelets (mean±SD, n=3-4).

Circulating Chilled Platelets have Hemostatic Function in CR3-DeficientMice.

Despite their rapid clearance in wild type mice, CM-Orange or CMFDAlabeled chilled platelets were functional 24 h after infusion intoCR3-deficient mice, as determined by three independent methods. First,chilled platelets incorporate into platelet aggregates in shed bloodemerging from a standardized tail vein bleeding wound (FIG. 6A-6E).CMFDA-positive room temperature platelets transfused into wild type mice(FIG. 6 b) and CNIFDA-positive chilled platelets transfused intoCR3-deficient mice (FIG. 6 d) formed aggregates in shed blood to thesame extent as CMFDA-negative platelets of the recipient mouse. Second,as determined by platelet surface exposure of the fibrinogen-bindingsite on α_(IIb)β₃ 24 hours after transfusion of CM-Orange-labeledchilled and rewarmed platelets into CR3 deficient mice following ex vivostimulation by thrombin. Third, CM-Orange platelets chilled and rewarmedwere fully capable of upregulation of P-selectin in response to thrombinactivation (FIG. 6 e).

Discussion

Cold-Induced Platelet Shape Change Alone does not Lead to PlateletClearance In Vivo

Cooling rapidly induces extensive platelet shape changes mediated byintracellular cytoskeletal rearrangements (Hoffmeister et al., 2001;White and Krivit, 1967; Winokur and Hartwig, 1995). These alterationsare partially but not completely reversible by rewarming, and rewarmedplatelets are more spherical than discoid. The idea that preservation ofplatelet discoid shape is a major requirement for platelet survival hasbeen a dogma, despite evidence that transfused murine and baboonplatelets activated ex vivo by thrombin circulate normally withextensive shape changes (Berger et al., 1998; Michelson et al, 1996).Here we have shown that chilling leads to specific changes in theplatelet surface that mediate their removal independently of shapechange, and that the shape change per se does not lead to rapid plateletclearance. Chilled and rewarmed platelets, preserved as discs withpharmacological agents, clear with the same speed as untreated chilledplatelets, and misshapen chilled and rewarmed platelets circulate likeroom temperature maintained platelets in CR3-deficient mice. The smallsize of platelets may allow them to remain in the circulation, escapingentrapment despite these extensive shape deformities.

Receptors Mediating Clearance of Chilled Platelets: CR3 and GP1bα

The normal platelet life span in humans is approximately 7 days (Aas,1958; Ware et 2000). The incorporation of platelets into small bloodclots engendered by continuous mechanical stresses undoubtedlycontributes to platelet clearance, because massive clotting reactions,such as occur during disseminated intravascular coagulation, causethrombocytopenia (Seligsohn, 1995). The fate of platelets in suchclotting reactions differs from that of infused ex vivo-activatedplatelets such as in the experiments of Michelson et al (Michelson etal., 1996) and Berger et al (Berger et al., 1998), because in vivoplatelet stimulation occurs on injured vessel walls, and the activatedplatelets rapidly sequester at these sites.

Isoantibodies and autoantibodies accelerate the phagocytic removal ofplatelets by Fc-receptor-bearing macrophages in individuals sensitizedby immunologically incompatible platelets or in patients with autoimmunethrombocytopenia, but otherwise little information exists regardingmechanisms of platelet clearance. We showed, however, that thequantities of IgG or IgM bound to chilled or room-temperature humanplatelets are identical, implying that binding of platelet-associatedantibodies to Fc-receptors does not mediate the clearance of cooledplatelets. We also demonstrated that chilling of platelets does notinduce detectable phosphatidylserine (PS) exposure on the plateletsurface in vitro militating against PS exposure and the involvement ofscavenger receptors in the clearance of chilled platelets.

Although many publications have referred to effects of cold on plateletsas “activation”, aside from cytoskeletally-mediated shape changes,chilled platelets do not resemble platelets activated by stimuli such asthrombin or ADP. Normal activation markedly increases surface P-selectinexpression, a consequence of secretion from intracellular granules(Berman et al., 1986). Chilling of platelets does not lead toup-regulation of P-selectin (Table 1), but the clearance of chilledplatelets isolated from wild-type or P-selectin-deficient mice isequally rapid (Berger et al., 1998). Activation also increases theamount of α_(IIb)β₃-integrin and its avidity for fibrinogen (Shattil,1999), but cooling does not have these effects (Table 1). The normalsurvival of thrombin-activated platelets is consistent with ourfindings.

We have shown that CR3 on liver macrophages is primarily responsible forthe recognition and clearance of cooled platelets. The predominant roleof CR3 bearing macrophages in the liver in clearance of chilledplatelets despite abundant CR3-expressing macrophages in the spleen isconsistent with the principally hepatic clearance of IgM-coatederythrocytes (Yan et al., 2000) and may reflect blood filtrationproperties of the liver that favor binding and ingestion by macrophageCR3. The extracellular domain of GP1bα binds avidly to CR3, and undershear stress in vitro supports the rolling and firm adhesion of THP-1cells (Simon et al., 2000). Cleavage of the extracellular domain ofmurine GP1bα results in normal survival of chilled platelets transfusedinto mice. GP1bα depletion of human chilled platelets greatly reducesphagocytosis of the treated platelets by macrophage-like cells in vitro.We propose, therefore, that GP1bα is the co-receptor for livermacrophage CR3 on chilled platelets leading to platelet clearance byphagocytosis.

The normal clearance of cold platelets lacking the N-terminal portion ofGP1bα rules out the many other CR3-binding partners, including moleculesexpressed on platelet surfaces as candidates for mediating chilledplatelet clearance. These ligand candidates include ICAM-2, fibrinogenbound to the platelet integrin α_(IIb)β₃, iC3b, P-selectin,glucosaminoglycans, and high molecular weight kininogen. We excludeddeposition of the opsonic C3b fragment iC3b as a mechanism for chilledplatelet clearance using mice deficient in complement factor 3, and theexpression level of α_(IIb)β₃ and fibrinogen binding are also unchangedafter chilling of platelets.

Binding to Activated vWf and Cold-Induced Binding to CR3 Appear to beSeparate Functions of GP1bα

GP1bα on the surface of the resting discoid platelet exists in lineararrays (FIG. 5A-5C) in a complex with GP1bα, GP1X and V, attached to thesubmembrane actin cytoskeleton by filamin-A and Filamin B (Stossel etal., 2001). Its role in hemostasis is to bind the activated form of vWfat sites of vascular injury. GP1bα binding to activated vWf isconstitutive and requires no active contribution from the platelet,since activated vWf binds equally well to GP1bα on resting or onstimulated platelets. Stimulation of platelets in suspension by thrombinand other agonists causes GP1bα to redistribute in part from theplatelet surface into an internal membrane network, the open canalicularsystem, but does not lead to platelet clearance in vivo (Berger et al.,1998; Michelson et al., 1996) or to phagocytosis in vitro (unpublishedobservations). Cooling of platelets however, causes GP1bα clusteringrather than internalization. This clustering is independent of barbedend actin assembly, because it occurs in the presence of cytochalasin B.

Despite cold's promoting recognition of platelet GP1bα by CR3, it has noeffect on interaction between GP1bα and activated vWf in vitro, andchilled platelets transfused into vWf-deficient mice disappear asrapidly as in wild-type mice. The separability of GP1bα's interactionwith vWf and CR3 suggests that selective modification of GP1bα mightinhibit cold-induced platelet clearance without impairment of GP1 bα'shemostatically important reactivity with vWf. Since all tests ofplatelet function of cooled platelets in vitro and after infusion intoCR3-deficient mice yielded normal results, suitably modified plateletswould predictably be hemostatically effective.

Physiological Importance of Cold-Induced Platelet Clearance.

Although gross platelet shape changes become obvious only attemperatures below 15° C., accurate biochemical analyses show thatcytoskeletal alterations and increased responsiveness to thrombin aredetectable as the temperature falls below 37° C. (Faraday and Rosenfeld,1998; Hoffmeister et al., 2001; Tablin et al., 1996). We refer to thosechanges as “priming” because of the many functional differences thatremain between cold-exposed and thrombin- or ADP-stimulated platelets.Since platelet activation is potentially lethal in coronary and cerebralblood vessels subjected to core body temperatures, we have proposed thatplatelets are thermosensors, designed to be relatively inactive at thecore body temperature of the central circulation but to become primedfor activation at the lower temperatures of external body surfaces,sites most susceptible to bleeding throughout evolutionary history(Hoffmeister et al., 2001). The findings reported here suggest thatirreversible changes in GP1 bα are the reason for the clearance ofcooled platelets. Rather than allowing chilled platelets to circulate,the organism clears low temperature-primed platelets by phagocytosis.

A system involving at least two clearance pathways, one for removal oflocally activated platelets and another for taking out excessivelyprimed platelets (FIG. 7), can possibly explain why chilled plateletscirculate and function normally in CR3-deficient mice and have aslightly prolonged circulation following removal of GP1bα. We proposethat some primed platelets enter microvascular clots on a stochasticbasis. Others are susceptible to repeated exposure to body surfacetemperature, and this repetitive priming eventually renders theseplatelets recognizable by CR3-bearing liver macrophages. Plateletsprimed by chilling are capable of normal hemostatic function inCR3-deficient mice, and coagulation contributes to their clearance.However, the slightly shorter survival time of autologous platelets inCR3-deficient mice examined is probably not ascribable to increasedclearance of normally primed platelets in microvascular clots, becausethe clearance rate of refrigerated platelets was indistinguishable fromthat of platelets kept at room temperature.

REFERENCES FOR BACKGROUND OF THE INVENTION AND EXAMPLE 1

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Example 2 Implication of the α_(M)β₂ (CR3) Lectin Domain in ChilledPlatelet Phagocytosis

α_(M)|₂ (CR3) has a cation-independent sugar-binding lectin site,located “C-T” to its I-domain (Thornton et al, J. Immonol. 156,1235-1246, 1996), which binds to mannans, glucans andN-Acetyl-D-glucosamine (GlcNAc). Since CD16b/α_(M)β₂ membrane complexesare disrupted by β-glucan, N-Acetyl-D-galactosamine (GalNAc), andmethyl-α-mannoside, but not by other sugars, it is believed that thisinteraction occurs at the lectin site of the α_(M)β₂ integrin (CR3)(Petty et al, J. Leukoc. Biol. 54, 492-494, 1993; Sehgal et al, J.Immunol. 150, 4571-4580, 1993).

The lectin site of α_(M)β₂ integrin has a broad sugar specificity (Ross,R. Critical Reviews in Immunology 20, 197-222, 2000). Although sugarbinding to lectins is usually of low affinity, clustering can cause amore robust interaction by increasing avidity. The clustering of GP1bαfollowing cooling, as shown by electron microscopy, suggests such amechanism. The most common hexosamines of animal cells are D-glucosamineand D-galactosamine, mostly occurring in structural carbohydrates asGlcNAc and GalNAc, suggesting that the α_(M)β₂ integrin lectin domainmight also bind to mammalian glycoproteins containing carbohydrates thatare not covered by sialic acid. The soluble form of GP1bα, glycocalicin,has a carbohydrate content of 60% comprising N- as well asO-glycosidically linked carbohydrate chains (Tsuji et al, J. Biol. Chem.258, 6335-6339, 1983). Glycocalicin contains 4 potential N-glycosylationsites (Lopez, et al, Proc. Natl. Acad. Sci., USA 84, 5615-5619, 1987).The 45 kDa region contains two sites that are N-glycosylated (Titani etal, Proc Natl Acad Sci 16, 5610-5614, 1987). In normal mammalian cells,four common core structures of O-glycan can be synthesized. All of themmay be elongated, sialylated, fucosylated and sulfated to formfunctional carbohydrate structures. The N-linked carbohydrate chains ofGP1bα are of the complex-type and di-, tri- and tetra-antennarystructures (Tsuji et al, J. Biol. Chem. 258, 6335-6339, 1983). They aresialylated GalNAc type structures with an α(1-6)-linked fucose residueat the Asn-bound GlcNAc unit. There is a structural similarity ofAsn-linked sugar chains with the Ser/Thr-linked: i.e., their position isof a common Gal-GlcNAc sequence. Results suggested that removal ofsialic acid and galactose has no influence on the binding of vWf toglycocalicin, but partial removal of GlcNac resulted in the inhibitionof vWf binding (Korrel et al, FEBS Lett 15, 321-326, 1988). A morerecent study proposed that the carbohydrate patterns are involved inmaintaining an appropriate functional conformation of the receptor,without participating directly in the binding of vWf (Moshfegh et al,Biochem. Biophys. Res. Communic. 249, 903-909, 1998).

A role of sugars in the interaction between chilled platelets andmacrophages has the important consequence that covalent modification,removal or masking of oligosaccharide residues could prevent thisinteraction. We hypothesized that if such prevention does not impairnormal platelet function, we may be able to modify platelets and enablecold platelet storage. Here, we show evidence that favor thishypothesis: 1) Saccharides inhibited phagocytosis of chilled plateletsby macrophages in vitro, and the specific sugars that are effectiveimplicated β-glucans as the relevant targets. Low concentrations ofβ-GlcNAc were surprisingly effective inhibitors, consistent with theidea that interference with a relatively small number of clusteredsugars may be sufficient to inhibit phagocytosis. Addition of sugars atconcentrations that maximally inhibited phagocytosis of chilledplatelets has no effect on normal GP1bα function (vWf-binding); 2) Aβ-GlcNAc-specific lectin, but not other lectins, bound avidly to chilledplatelets; 3) Removal of GP1bα or β-GlcNAc residues from plateletsurfaces prevented this binding (since β-GlcNAc removal exposed mannoseresidues, it did not prevent phagocytosis by macrophages which havemannose receptors); 4) Blocking of exposed β-Glucans on chilledplatelets by enzymatic addition of galactose markedly inhibitedphagocytosis of chilled platelets by macrophages in vitro and extendedthe circulation times of chilled platelets in normal animals.

Effect of Monosaccharides on Phagocytosis of Chilled Platelets.

To analyze the effects of monosaccharides on platelet phagocytosis,phagocytes (differentiated monocytic cell line THP-1) were incubated inmonosaccharide solutions at various concentrations, and the chilled orroom temperature platelets were added. Values in the Figures aremeans±SD of 3-5 experiments comparing percentages of orange-positivemonocytes containing ingested platelets incubated with RT or chilledplatelets). While 100 mM D-glucose inhibited chilled plateletphagocytosis by 65.5% (P<0.01), 100 mM D-galactose did not significantlyinhibit chilled platelet phagocytosis (n=3) (FIG. 8A). The D-glucoseα-anomer (α-glucoside) did not have an inhibitory effect on chilledplatelet phagocytosis, although 100 mM inhibited by 90.2% (FIG. 8B) Incontrast, β-glucoside inhibited phagocytosis in a dose-dependent manner(FIG. 8B). Incubation of the phagocytes with 100 mM β-glucosideinhibited phagocytosis by 80% (p<0.05) and 200 mM by 97% (P<0.05),therefore we concluded that the β-anomer is preferred. D-mannose and itsα- and β-anomers (methyl-α-D-mannopyranoside (FIG. 8C) andmethyl-β-D-mannopyranoside (FIG. 8C) had no inhibitory effect on chilledor RT platelet phagocytosis. Incubation of phagocytes using 25 to 200 mMGlcNAc (N-acetyl-D-glucosamine) significantly inhibited chilled plateletphagocytosis. Incubation with 25 mM GlcNac was sufficient to inhibit thephagocytosis of chilled platelets by 86% (P<0.05) (FIG. 8D), whereas 10μM of the β-anomer of GlcNAc inhibited the phagocytosis of chilledplatelets by 80% (p<0.01) (FIG. 8D). None of the monosaccharides had aninhibitory effect on RT platelet phagocytosis. Table 2 summarizes theinhibitory effects of monosaccharides at the indicated concentrations onchilled platelet phagocytosis (**P<0.01, *P<0.05). None of themonosaccharides inhibited thrombin or ristocetin induced human plateletaggregation or induced α-granule secretion as measured by P-selectinexposure.

TABLE 2 Inhibitory effects of monosaccharides on chilled plateletphagocytosis Monosaccharides % inhibition phagocytosis mM D-(+)-glucose65.5  100 D-(+)-galactose — 100 Methyl-α-D- 90.2* 100 glucopyranosideMethyl-β-D- 80.2* 100 gludopyranoside 97.1* 200 D-(+)-mannose — 100Methyl-α-D- — 100 mannopyranoside Methyl-β-D- — 100 mannopyranosideβ-GlcNac 80.9* 0.01 GlcNac 86.3* 25 83.9* 100 83.1* 200

Binding of Various Lectins to Room Temperature Platelets or ChilledPlatelets.

β-GlcNAc strongly inhibited chilled human platelet phagocytosis in vitroat μM concentrations, indicating that GlcNac is exposed after incubationof platelets in the cold. We then investigated whether wheat germagglutinin (WGA), a lectin with specificity towards the terminal sugar(GlcNAc), binds more effectively to chilled platelets than to roomtemperature platelets. Washed, chilled or room temperature plateletswere incubated with 2 μg/ml of FITC coupled WGA or FITC coupledsuccinyl-WGA for 30 min at room temperature and analyzed by flowcytometry. FIGS. 9A and 9B show the dot plots after incubation withFITC-WGA of room temperature (RT) or chilled (Cold) human platelets. WGAinduces platelet aggregation and release of serotonin or ADP atconcentrations between 25-50 g/ml WGA (Greenberg and Jamieson, Biochem.Biophys. Acta 345, 231-242, 1974). Incubation with 2 μg/ml WGA inducedno significant aggregation of RT-platelets (FIG. 9A, RT w/WGA), butincubation of chilled platelets with 2 μg/ml WGA induced massiveaggregation (FIG. 9B, Cold/w WGA). FIG. 9C shows the analysis ofFITC-WGA fluorescence binding to chilled or room temperature platelets.To verify that the increase of fluorescence binding is not aggregationrelated, we used succinyl-WGA (S-WGA), a dimeric derivate of the lectinthat does not induce platelet aggregation (Rendu and Lebret, Thromb Res36, 447-456, 1984). FIGS. 9D and 9E show that succinyl-WGA (S-WGA) didnot induce aggregation of room temperature or chilled platelets, butresulted the same increase in WGA binding to chilled platelets versusroom temperature platelets (FIG. 9F). The enhanced binding of S-WGAafter chilling of platelets cannot be reversed by warming of chilledplatelets to 37° C.

Exposed β-GlcNAc residues serve as substrate for aβ1,4glactosyltransferase enzyme that catalyses the linkageGalβ-1GlcNAcβ1-R. In support of this prediction, masking of β-GlcNAcresidues by enzymatic galactosylation inhibited S-WGA binding to coldplatelets, phagocytosis of chilled platelets by THP-1 cells, and therapid clearance of chilled platelets after transfusion into mice. Theenzymatic galactosylation, achieved with bovineβ1,4galactosyltransferase and its donor substrate UDP-Gal, decreasedS-WGA binding to chilled human platelets to levels equivalent to roomtemperature platelets. Conversely, the binding of the galactose-specificRCA I lectin increased by ˜2 fold after galactosylation. UDP-Glucose andUDP alone had no effect on S-WGA or RCA I binding to chilled or roomtemperature human platelets.

We found that the enzymatic galactosylation of human and mouse plateletsis efficient without addition of exogenous β1,4galactosyltransferase.The addition alone of the donor substrate UDP-Gal reduces S-WGA bindingand increases RCA I binding to chilled platelets, inhibits phagocytosisof chilled platelets by THP1 cells in vitro, and prolongs thecirculation of chilled platelets in mice. An explanation for thisunexpected finding is that platelets reportedly slowly releaseendogenous galactosyltransferase activity. A least one form ofβ1,4galactosyltransferases, β4Gal T1, is present in human plasma, onwashed human platelets and in the supernatant fluids of washedplatelets. Galactosyltransferases may associate specifically with theplatelet surface. Alternatively, the activity may be plasma-derived andleak out of the platelet's open canalicular system. In either case,modification of platelet glycans responsible for cold-mediated plateletclearance is possible by simple addition of the sugar-nucleotide donorsubstrate, UDP-Gal.

Importantly, both chilled and non-chilled platelets show the sameincrease in RCA I binding after galactosylation, implying that β-GlcNAcresidues are exposed on the platelet surface independent of temperature.However chilling is a requirement for recognition of β-GlcNAc residuesby S-WGA and by the α_(M)β₂ integrin. We have demonstrated that chillingof platelets induces an irreversible clustering of GP1b. Generallylectin binding is of low affinity and multivalent interactions with highdensity of carbohydrate ligands increases binding avidity. Possibly thelocal densities of exposed β-GlcNAc on the surface of non-chilledplatelets are too low for recognition, but cold-induced clustering ofGP1bα provides the necessary density for binding to S-WGA or the α_(M)β₂integrin lectin domain. We confirmed by S-WGA and RCA-I binding flowcytometry that UDP-Gal transfers galactose onto murine platelets in thepresence or absence of added galactosyl transferase and documented thatchilled, galactosylated murine platelets circulate and initially survivesignificantly better than untreated room temperature platelets.

Although the earliest recoveries (<2 min) did not differ betweentransfused RT, chilled and chilled, galactosylated platelets,galactosylation abolished an initial platelet loss of about 20%consistently observed with RT platelets.

Galactosylation of murine and human platelets did not impair theirfunctionality in vitro as measured by aggregation and P-selectinexposure induced by collagen related peptide (CRP) or thrombin atconcentrations ranging from maximally effective to three orders ofmagnitude lower. Importantly, the aggregation responses of unmodifiedand galactosylated chilled human platelets to a range of ristocetinconcentrations, a test of the interaction between GP1b and activatedVWF, were indistinguishable or slightly better. The attachment pointsfor N-linked glycans on GP1bα are outside of the binding pocket for VWF.Moreover, mutant GP1bα molecules lacking N-linked glycans bind VFWtightly.

Using FITC labeled lectins with specificities towards β-galactose (R.communis lectin/RCA), 2-3 sialic acid (Maackida amurensis lectin/MAA) or2-6 sialic acid (Sambucus Nigra bark lectin/SNA), we could not detectincreased binding after chilling of platelets by flow cytometry (FIG.10), showing that exposure after chilling of platelets is restricted toGlcNAc.

We localized the exposed β-GlcNAc residues mediating α_(M)β₂ lectindomain recognition of GP1bα N-glycans. The extracellular domain of GP1bαcontains 60% of total platelet carbohydrate content in the form of N-and O-glycosidically linked carbohydrate chain. Accordingly, binding ofperoxidase-labeled WGA to GP1bα is easily detectable in displays oftotal platelet proteins resolved by SDS-PAGE, demonstrating that GP1bαcontains the bulk of the β-GlcNAc-residues on platelets, and binding ofWGA to GP1bα is observable in GP1bα immunoprecipitates. UDP-Gal with orwithout added galactosyltransferase diminishes S-WGA binding to GP1bα,whereas RCA I binding to GP1bα increases. These findings indicate thatgalactosylation specifically covers exposed β-GlcNAc residues on GP1bα.Removal of the N-terminal 282 residues of GP1bα from human plateletsurfaces using the snake venom protease mocarhagin, which inhibitedphagocytosis of human platelets by THP-1 cells in vitro, reduces S-WGAbinding to chilled platelets nearly equivalent to S-WGA room temperaturebinding levels. WGA binds predominantly to the N-terminus of GP1bαreleased by mocarhagin into □platelet supernatant fluids as apolypeptide band of 45 kDa recognizable by the monoclonal antibody SZ2specific for that domain. The glycans of this domain are N-linked. Asmall portion of GP1bα remains intact after mocarhagin treatment,possibly because the open canalicular system of the platelet sequestersit. Peroxidase-conjugated WGA weakly recognizes the residual plateletassociated GP1bα C-terminus after mocarhagin cleavage, identifiable withmonoclonal antibody WM23.

The cold-induced increase in binding of human platelets to α_(M)β₂integrin and to S-WGA occurs rapidly (within minutes). The enhancedbinding of S-WGA to chilled platelets remained stable for up to 12 daysof refrigerated storage in autologous plasma. RCA I binding remainedequivalent to room temperature levels under the same conditions.Galactosylation doubled the binding of RCA I lectin to platelets andreduced S-WGA binding to baseline RT levels. The increase in RCA I anddecrease in S-WGA binding were identical whether galactosylationproceeded or followed storage of the platelets in autologous plasma forup to 12 days. These findings indicate that galactosylation of plateletsto inhibit lectin binding is possible before or after refrigeration andthat the glycan modification is stable during storage for up to 12 days.Platelets stored at room temperature rapidly lose responsiveness toaggregating agents; this loss does not occur with refrigeration.Accordingly, refrigerated platelets with or without galactosylation,before or after storage, retained aggregation responsiveness to thrombinfor up to 12 days of cold storage.

Effects of β-hexosaminidase (β-Hex) and Mocarhagin (MOC) on FITC-WGALectin Binding to Chilled Versus Room Temperature Stored Platelets.

The enzyme β-hexosaminidase catalyzes the hydrolysis of terminalβ-D-N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) residuesfrom oligosaccharides. To analyze whether removal of GlcNAc residuesreduces the binding of WGA to the platelet surface, chilled and roomtemperature washed human platelets were treated with 100 U/ml β-Hex for30 min at 37° C. FIG. 11A shows the summary of FITC-WGA binding to thesurface of room temperature or chilled platelets obtained by flowcytometry before and after β-hexosaminidase treatment. FITC-WGA bindingto chilled platelets was reduced by 85% after removal of GlcNac (n=3).We also checked whether, as expected, removal of GP1bα from the plateletsurface leads to reduced WGA-binding after platelet chilling. GP1bα wasremoved from the platelet surface using the snake venom mocarhagin(MOC), as described previously (Ward et al, Biochemistry 28, 8326-8336,1996). FIG. 11B shows that GP1bα removal from the platelet surfacereduced FITC-WGA binding to chilled platelets by 75% and had littleinfluence on WGA-binding to GP1bα-depleted room temperature platelets(n=3). These results indicate that WGA binds mostly to oligosaccharideson GP1bα after chilling of human platelets, and it is very tempting tospeculate that the Mac-1 lectin site also recognizes these exposedsugars on GP1bα leading to phagocytosis.

Masking of Human Platelet GlcNAc Residues by Galactose-Transfer GreatlyReduces their Phagocytosis after Chilling In Vitro and DramaticallyIncreases their Survival in Mice.

To achieve galactose transfer onto platelets, isolated human plateletswere incubated with 200 μM UDP-galactose and 15 mU/ml galactosetransferase for 30 min at 37° C., followed by chilling or maintenance atroom temperature for 2 h. Galactosylation reduced FITC-WGA bindingalmost to resting room temperature levels. Platelets were fed to themonocytes and platelet phagocytosis was analyzed as described above.FIG. 12 shows that galactose transfer onto platelet oligosaccharidesreduces greatly chilled platelet (Cold) phagocytosis, but does notaffect the phagocytosis of room temperature (RT) platelets (n=3). Theseresults show that in vitro the phagocytosis of chilled platelets can bereduced through coverage of exposed GlcNAc residues. We tested whetherthis approach could be extended to animals and used to increase thecirculation time of chilled platelets. Murine platelets were isolatedand stained with CMFDA. Using the same approach of galactose transferdescribed for human platelets above, wild type murine platelets weregalactosylated and chilled, or not, for 2 hours. 10⁸ Platelets weretransfused into wild type mice and their survival determined. FIG. 13shows the survival of these chilled, galactosylated murine plateletsrelative to untreated platelets. Both platelets kept at room temperature(RT) and the galactosylated chilled platelets (Cold+GalT) had almostidentical survival times, whereas chilled untreated platelets (Cold)were cleared rapidly as expected. We believe galactosylated chilledplatelets will circulate in humans.

We noted that our control reaction, in which galactose transferase washeat-inactivated also resulted in glycan modification of platelets asoccurred in the experimental reaction with active galactose transferase,as judged by WGA binding (FIG. 14A), in vitro phagocytosis results inhuman platelets (FIG. 14B), and survival of murine platelets (FIG. 14C).Therefore, we conclude that platelets contain galactose transferaseactivity on their surface, which is capable of directing to glycanmodification using only UDP-galactose without the addition of anyexogenous galactose transferase. Thus, glycan modification of plateletscan be achieved simply by incubation with UDP-galactose.

UDP-Galactose Incorporate into Human Platelets in a Time DependentMatter.

In another set of experiments we have shown that ¹⁴C-labeledUDP-galactose incorporates into human platelets in a time dependentmanner in the presence or absence of the enzyme galactosyl transferase.FIG. 15 shows the time course of ¹⁴C-labeled UDP-galactose incorporationinto washed human platelets. Human platelets were incubated with¹⁴C-labeled UDP-galactose for different time intervals in the absence ofgalactosyl transferase. The platelets were then washed and the ¹⁴Cradioactivity associated with platelets measured.

Example 3 Enzymatic Modification of Platelet β-Glycans InhibitPhagocytosis of Cooled Platelets by Macrophages In Vitro and AccommodateNormal Circulation In Vivo

Our preliminary experiments have demonstrated the enzymatic covering ofGlcNAc residues on GP1bα using galactose-transfer (glycan modification)onto chilled human platelet surfaces greatly reduced their in vitrophagocytosis. One interpretation of these findings is that GP1bαstructure is altered on the surface of chilled human and murineplatelets. This causes the exposure or clustering of GlcNAc, which isrecognized by the lectin binding domain of αMβ2 leading to plateletremoval β-GlcNAc exposure can be measured by WGA binding and possibly bybinding of recombinant αMβ2 lectin domain peptides. Resting humanplatelets bind WGA, which increases greatly after chilling. We proposethat galactose transfer (glycan modification) will prevent GP1bα'sinteraction with αMβ2-lectin but not with vWf. This modification(galactose transfer onto platelet surface) leads to normal survival ofchilled platelets in WT mice as shown by our preliminary experiments.

Example 4

This example shows that the αMβ2 lectin site mimics WGA and sugarmodifications prevent the engagement of the recombinant lectin site withchilled platelets. Dr. T. Springer (Corbi, et al., J. Biol Chem. 263,12403-12411, 1988) provided the human αM cDNA and several anti-αMantibodies. The smallest r-huαM construct exhibiting lectin activitythat has been reported includes its C-T and a portion of its divalentcation binding region (residues 400-1098) (Xia et al, J Immunol 162,7285-7293, 1999). The construct is 6×His-tagged for ease ofpurification. We first determined if the recombinant lectin domain canbe used as a competitive inhibitor of chilled platelet ingestion in thephagocytic assay. Competition proved that the αM lectin site mediatesbinding to the platelet surface and initiates phagocytosis. As controls,a construct lacking the lectin-binding region of αM was used and therecombinant protein was denatured. Lectin binding domain functions as aspecific inhibitor of chilled platelet ingestion. We made a αM constructthat include GFP and express and labeled the αM-lectin binding site withFITC and used it to label the surface of chilled platelets by flowcytometry. Platelets were labeled with CMFDA. We found that chilledplatelets bind more efficiently to the αM lectin side of αMβ2 integrincompared to room temperature platelets. The lectin side and wholeαM-construct (Mac-1) was expressed in Sf9 insect cells.

The platelet sugar chains are modified to inhibit theplatelet-oligosaccharide interaction with the r-huαM-lectin site. Theefficiency of sugar modifications is also monitored by inhibition of thebinding of fluorescent-lectin domain binding to platelets by flowcytometry.

The recovery and circulation times of room temperature, chilled andchilled-modified platelets are compared to establish that galactosetransfer onto chilled murine platelets results in longer circulatingplatelets. Room temperature, chilled and chilled-modified platelets arestained with CMFDA, and 10⁸ platelets transfused into wild type mice asdescribed above. The mice are bled immediately (<2 min.), 30 min, 1 h,2, 24, 48 and 72 hours after transfusion. The blood obtained is analyzedusing flow cytometry. The percentage of fluorescent labeled plateletswithin the gated platelet population measured immediately afterinjection is set as 100%. The recovery of fluorescently labeledplatelets obtained at the various time points is calculated accordingly.

Example 5

This example demonstrates that chilled, unmodified and chilled,galactosylated (modified) platelets have hemostatic function in vitroand in vivo. Chilled platelets are not “activated” in the sense ofagonist-stimulated platelets. Patients undergoing surgery underhypothermic conditions may develop thrombocytopenia or show severehemostatic post-operative impairments. It is believed that under thesehypothermic conditions, platelets might lose their functionality.However, when patients undergo hypothermic surgery, the whole organismis exposed to hypothermia leading therefore to changes in multipletissues. Adhesion of non-chilled platelets to hepatic sinusoidalendothelial cells is a major mechanism of cold preservation injury(Takeda, et al. Transplantation 27, 820-828, 1999). Therefore, it islikely that it is the interaction between cold hepatic endothelium andplatelets, not platelet chilling per se, that leads to deleteriousconsequences under hypothermic conditions of surgery or trans-plantationof cold preserved organs (Upadhya et al, Transplantation 73, 1764-1770,2002). Two approaches showed that chilled platelets have hemostaticfunction. In one approach, the circulation of chilled platelets inαMβ2-deficient mice facilitates studies of platelet function aftercooling. In the other approach, the function of modified chilled and(presumably) circulating platelets was tested.

Human and murine unmodified and modified (galactosylated) chilledplatelets were tested for functionality, including in vitro aggregationto agonists, P-selectin exposure and fibrinogen binding.

αMβ2 deficient or WT mice are transfused with murine chilled/RTplatelets modified or not, and allowed to circulate for 30 min., 2 and24 hours. We determine if chilled platelets contribute to clottingreactions caused by tail vein bleeding and if these platelets bindagents such as fibrinogen after activation. We also determine howchilled platelets, modified or not, contribute to clotting on ferricchloride injured and exteriorized mouse mesenteries, an in vivothrombus-formation model that we developed. This method detects thenumber of platelets adherent to injured vessels and has documentedimpaired platelet vessel wall interactions of platelets lackingglycoprotein V or β3-integrin function (Ni et al., Blood 98, 368-3732001; Andre, et al. Nat Med 8, 247-252, 2002). Last, we determine thestorage parameters of the modified platelets.

In vitro platelet function is compared using aggregation with thrombinand ADP and botrocetin induced vWf-binding to murine platelets. Murineand human chilled platelets modified (galactosylated) or unmodifiedplatelets are normalized to a platelet concentration of 0.3×10⁹/mm³, andaggregation induced using the various agonists according to standardprotocols (Bergmeier, et al. 2001 276, 25121-25126, 2001). To studyvWf-binding we activate murine vWf using botrocetin and analyze thebinding of fluorescently labeled vWf to chilled platelets modified ornot in PRP (Bergmeier, et al. 2001 276, 25121-25126, 2001). To evaluatewhether degranulation of platelets occurs during modification, we alsomeasure P-selectin exposure of chilled murine and human plateletsmodified or not using fluorescent labeled anti-P-selectin antibodies byflow cytometry (Michelson et al., Proc. Natl. Acad. Sci., USA 93,11877-11882, 1996).

10⁹ CMFDA-labeled platelets are transfused into mice, first verifyingthat these platelets are functional in vitro. We determine whetherchilled platelets contribute to aggregation by transfusing chilled orroom temperature CMFDA-labeled platelets into αMβ2 deficient mice. At 30min., 2 hours and twenty-four hours after the infusion of platelets, astandard tail vein bleeding test is performed (Denis, et al. Proc NatlAcad Sci USA 95, 9524-9529, 1998). The emerging blood is fixedimmediately in 1% formaldehyde and platelet aggregation is determined bywhole blood flow cytometry. Platelet aggregates appear as bigger sizedparticles in the dot plot analysis. To verify that the transfusedplatelets do not aggregate in the normal circulation we also bleed themice through the retroorbital eye plexus into an anticoagulant.Platelets do not form aggregates under these bleeding conditions. Theemerging blood is fixed immediately and platelets are analyzed by flowcytometry in whole blood as described above. Platelets are identifiedthrough binding of a phycoerythrin-conjugated α_(IIb)β₃ specificmonoclonal antibody. The infused platelets in the blood sample areidentified by their CMFDA-fluorescence. Non-infused platelets areidentified by their lack of CMFDA fluorescence (Michelson, et al, Proc.Natl. Acad. Sci., U.S.A. 93, 11877-11882, 1996). The same set of testsis performed with CMFDA modified (galactosylated) chilled plateletstransfusing these platelets into αMβ2 and WT. This experiment testsaggregation of chilled platelets modified or not in shed blood.

10⁹ CM-orange labeled unmodified chilled or room temperature plateletsare transfused into αMβ2 deficient mice to verify that these plateletsare functional in vitro. At 30 min., 2 h and twenty-four hours after theinfusion of CM-orange labeled platelets, PRP is isolated as describedand analyzed by flow cytometry. P-selectin exposure is measured using ananti FITC-conjugated anti P-selectin antibody (Berger, et al, Blood 92,4446-4452, 1998). Non-infused platelets are identified by their lack ofCM-orange fluorescence. The infused platelets in the blood sample areidentified by their CM-orange fluorescence. CM-orange and P-selectinpositive platelets appear as double positive fluorescently(CM-orange/FITC) stained platelets. To verify that chilled plateletsstill expose P-selectin after thrombin activation, PRP is activatedthrough the addition of thrombin (1 U/ml, 2 min at 37° C.) andP-selectin exposure is measured as described. To analyze the binding offibrinogen to α_(IIb)β₃, isolated platelets are activated through theaddition of thrombin (1 U/ml, 2 min, 37° C.) and Oregon-green coupledfibrinogen (20 μg/ml) added for 20 min at 37° C. (Heilmann, et al,Cytometry 17, 287-293, 1994). The samples are analyzed immediately byflow cytometry. The infused platelets in the PRP sample are identifiedby their CM-orange fluorescence. CM-orange and Oregon-green positiveplatelets appear as double positive fluorescently stained(CM-orange/Oregon green) platelets. The same sets of experiments areperformed with CM-orange labeled modified (galactosylated) chilledplatelets transfused into αMβ2 deficient and WT mice.

Example 6 In Vivo Thrombosis Model

First, we show the delivery of RT and unmodified chilled platelets toinjured endothelium of αMβ2 deficient mice using double fluorescentlylabeled platelets. The resting blood vessel is monitored for 4 min.,then ferric chloride (30 μl of a 250-mM solution) (Sigma, St Louis, Mo.)is applied on top of the arteriole by superfusion, and video recordingresumed for another 10 min. Centerline erythrocyte velocity (Vrbc) ismeasured before filming and 10 min after ferric chloride injury. Theshear rate is calculated on the basis of Poiseuille's law for aNewtonian fluid (Denis, et al, Proc Natl Acad Sci USA 95, 9524-9529,1998). These experiments show if chilled platelets have normalhemostatic function. We repeat these experiments in WT mice comparing RTand galactosylated chilled platelets using two different, fluorescentlylabeled platelet populations injected into the same mouse and analyzethe thrombus formation and incorporation of both platelet populations.

We then compare in vitro platelet functions and survival and in vivohemostatic activity of chilled and modified chilled murine plateletsstored for 1, 5, 7 and 14 days under refrigeration as described above.We compare the recovery and circulation times of these stored chilledand modified chilled platelets and prove that: 1) the modificationthrough galactose transfer onto chilled murine platelets is stable afterthe long term refrigeration; and 2) that these platelets functionnormally. Survival experiments are performed as described above. We useWGA binding, to verify that GlcNAc residues remain covered by galactoseafter the longer storage time points. As an ultimate test that thesemodified, stored platelets are functionally intact and contribute tohemostasis, we transfuse them into total-body-irradiated mice (Hoyer, etal, Oncology 49, 166-172, 1992). To obtain the sufficient numbers ofplatelets, we inject mice with commercially available murinethrombopoietin for seven days to increase their platelet count (Lok, etal Nature 369, 565-558, 1994). Isolated platelets are modified using theoptimized galactose transfer protocol, stored under refrigeration,transfused, and tail vein bleeding times measured. Since unmodifiedchilled platelets do not persist in the circulation, a comparison ofmodified cooled platelets with room temperature stored platelets is notnecessary at this point. The murine platelets are stored underrefrigeration in standard test tubes. If a comparison with roomtemperature stored murine platelets is necessary we switch to primateplatelets. Rather than engineer special down-scale, gas-permeablestorage containers to accommodate mouse platelets, such comparisons aremore appropriate for primates (including humans) for which roomtemperature storage bags have been designed.

Example 7 Galactosylation of Platelets in a Platelet Concentrate

Four different platelet concentrates were treated with increasingconcentrations of UDP galactose: 400 μM, 600 μM, and 800 μM. Futureexperiments will use between 10 μM and 5000 μM UDP galactose. RCAbinding ratio measurements showed a dose dependent increase ingalactosylation in the four samples tested. (FIG. 16). Our resultsprovide evidence that galactosylation is possible in plateletconcentrates.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit and scope ofthe invention. It is intended to encompass all such modifications withinthe scope of the appended claims. All references, patents and patentpublications that are recited in this application are herebyincorporated by reference herein in their entirety.

Example 8 Evaluation of the In Vivo Survival of UDP-Galactose TreatedPlatelets Stored in the Cold

The technology for galactosylating human platelets with the use of theactivated carbohydrate substrate UDP-galactose may allow large-scalehuman platelet storage under refrigeration (4° C.). Untreated plateletsstored at 4° C. are rapidly cleared from the circulation. In contrast,untreated platelets stored at room temperature survive for ˜5-7 daysfollowing transfusion. The present study is intended to demonstrate thatthe galactosylated modified human platelets circulate in vivo wheninfused autologously into individuals.

The reason for the removal of chilled platelets from the circulation hasrecently been defined. Cooling of platelets causes clustering of theplatelet GPIb/V/IX complex on the platelet surface. The αMβ2 integrinreceptor (CR3, Mac-1) present on hepatic macrophages recognizesclustered GPIbβ molecules, and platelets are ingested by themacrophages. The αMβ2 integrin receptor contains a carbohydrate bindingdomain (lectin domain) that is critical for the recognition of exposedβ-N-acetylglucosamine (βGlcNAc) residues on the platelet surface bymacrophages. Covering of exposed βGlcNAc residues by enzymaticgalactosylation prevents recognition and phagocytosis of chilledplatelets. This has been extensively demonstrated in a mouse model,where chilled and galactosylated murine platelets have survival superiorto that of room temperature stored platelets. In vitro studies usinghuman platelets indicate that galactosylated platelets stored at 4° C.are likely also to circulate when transfused into humans.

To determine and demonstrate that galactosylated modified humanplatelets survive and circulate in vivo when infused autologously intoindividuals. This will be determined by comparing the survival rates ofradiolabeled refrigerated (2°-8° C.) platelets with or withoutgalactosylation to radiolabeled non-galactosylated platelets stored atroom temperature (22°±2° C.) and in the cold (Stored for 36 to 48 hrs).

The following describes a Phase I study in which in vivo recovery andhalf-life of autologously-infused galactosylated platelets in normal,healthy volunteer group subjects is determined.

Six (6) healthy donors will donate a unit of apheresis platelets. Thecollected apheresis product will be divided into two bags. One bag willhave the platelets treated with UDP-galactose and stored underrefrigeration for 36-48 hours. The other platelet bag will either bestored under refrigeration or as per current FDA guidelines at roomtemperature for 36-48 hours. The two bags of platelets will each beradiolabeled with a different radioactive isotope, ⁵¹Chromium or¹¹¹Indium and 5-10 mL of labeled platelets will be injected in thehealthy volunteers. Blood samples will be drawn before and at 2 hoursafter the transfusion and then on days 1, 2, 3, 5, 7 and 10 afterreinfusion, and the post-transfusion recovery and survival of theplatelets will be determined.

The experimental material injected in the healthy volunteers will be5-10 mL aliquots of platelets that have been taken from the studysubjects, with or without modification by galactosylation and eitherstored at room temperature (22±2° C.) or stored in the cold (4±2° C.).

Upon confirmation of eligibility and enrollment in the study, healthydonors will be recruited to donate a unit of platelets on theHaemonetics MCS+ apheresis machine. This machine draws whole blood froma donor's arm, centrifuges the blood to separate the platelets from theplasma and the red cells, collects the platelets with a small amount ofplasma and returns most of the plasma and the red cells back to thedonor. The collected platelets and plasma will be divided into two bags.Each bag will be weighed and the platelet count determined on the day ofcollection, day 1 and day of infusion. After collection the plateletswill be rested for 1 hour. After the resting period one platelet bagwill be treated with a naturally occurring sugar substance,UDP-galactose. This bag will be incubated for 1 hour at 37° C. andstored under refrigeration. The other platelet bag will likewise beincubated for 1 hour at 37° C. and stored under refrigeration or as percurrent FDA guidelines at room temperature. On Day 1 followingcollection a sample from each bag will be sent to a microbiology lab forculture.

The platelet culture results will be recorded along with the results ofa gram stain sample that will be sent to the lab on the day ofreinfusion. If either report is positive the platelet units will not bereinfused. The two bags of platelets will each be radiolabeled with adifferent radioactive isotope, ⁵¹Chromium or ¹¹¹Indium. Blood sampleswill be drawn before and at 2 hours after and then on days 1, 2, 3, 5, 7and 10 after the reinfusion. The blood samples will be analyzed forradioactivity to determine the post-transfusion recovery and survival ofthe platelets. Since the two units of platelets have been tagged withdifferent radioactive isotopes, we will be able to distinguish betweenthe platelets that were subjected to the UDP Galactose and those thatare untreated.

UDP-galactose (Uridine-5′-diphosphogalactose) is a natural sugarcompound found in the human body. It is used in this study as a donorfor the addition of galactose to the surface of the human platelets tobe transfused. The UDP-galactose was manufactured by Roche DiagnosticsGmbH and is over 97% pure. It contains trace quantities of by-productsof the manufacturing process. It was formulated and filled into syringesby a licensed filling facility, and tested for sterility andpyrogenicity.

Blood samples taken from each study subject will be tested for plateletcount and anti-platelet antibodies before and at two weeks and threemonths after the platelet infusion.

Between 5 and 10 mL of platelets radiolabeled with the two differentradioactive isotope, ⁵¹Chromium or ¹¹¹Indium, will be injected at day 0.Blood samples will be drawn before and at 2 hours and on days 1, 2, 3,5, 7 and 10 after reinfusion.

During each reinfusion, the subject will be carefully monitored foradverse reactions, most usually fever, chills, dyspnea, urticaria orpain (infusion site, chest pain or other), or significant changes invital signs. In addition, each subject will be queried during the followup period visits up to three months after the infusion to obtaininformation on any occurrence of adverse events during that time.Non-modified and modified platelets will be characterized by a number ofin vitro analyses including but not limited to: pH, pO2, pCO2,bicarbonate, hypotonic shock response, morphology, extent of shapechange, ATP levels, glucose, O2 consumption, p-Selectin, and Annexin Vbinding.

REFERENCES Incorporated Herein in their Entirety

-   1. Becker, Tucecelli et al. G. Transfusion 13, 61 (1973).-   2. Hoffmeister, Felbinger et al. Cell 10, 87 (2003).-   3. Valeri, Ragno et al. Transfusion 44(6):865-70 (2004).-   4. Murphy S, Oski F A et al N Engl J Med. 1969 16; 281(16):857-62-   5. Dumont, VandenBroeke et al. Transfus Med Rev. 13(1):31-42 (1999).-   6. Michelson, Adelman et al. J Clin Invest. 81(6):1734-40 (1988).-   7. Ribeiro, Swann et al. Thromb Res. 66(6):619-27 (1992).-   8. Jaremo, Rubach-Dahlberg et al. Thromb Res. 69(5):467-77 (1993).-   9. Hoffmeister, Josefsson et al. Science September 12;    301(5639):1531-4 (2003).-   10. J Pediatr Gastroent Nutr 13:26 0-266 (1991).-   11. J Pediatr Gastroent Nutr 19:100-108 (1994).-   12. Mizoguchi, Ono et al., Eur J Pediatr 159: 851-853 (2000).-   13. Lancet 346:1073-1074 (1995).-   14. Acta Medica Scandinav Suppl 177:1-125 (1947).-   15. Lazarowski, Shea et al. Mol Pharmacol 63: 1190-1197 (2003).-   16. Josefsson et al J Biol Chem. 2005 Mar. 1; [Epub ahead of print]-   17. Puget Sound Blood Center SOP, “Radiolabeling Fresh Platelets    with ¹¹¹Indium Oxine or ⁵¹Chromium”, Rev. Jan. 12, 2005-   18. Puget Sound Blood Center SOP, “Radiolabeling Stored Apheresis    Platelets with ⁵¹Chromium”, Rev. Jan. 12, 2005-   19. Puget Sound Blood Center SOP, “Radiolabeling Stored Apheresis    Platelets with ¹¹¹Indium Oxine”, Rev. Jan. 12, 2005

What is claimed is:
 1. An apparatus for processing a sample ofplatelets, the apparatus comprising a first sterile container having oneor more ports including a first container port and containing apreparation of blood cells comprising platelets, a second sterilecontainer having one or more ports including a second container port andcontaining a blood cell modifying agent which comprises cytidine5′-monophospho-N-acetylneuraminic acid, the first container beingadapted to the second container through a first sterile conduitreversibly attachable to the first container port and the secondcontainer port, the first sterile conduit further comprising a valve,wherein the blood cell modifying agent present in the second sterilecontainer is introduced into the first sterile container and theplatelets in the preparation of blood cells therein are rendered coldstorage competent after the platelets are contacted with the blood cellmodifying agent.
 2. The apparatus of claim 1, further comprising a thirdsterile container having one or more ports including a third containerport adapted to the first container through a second sterile conduitreversibly attachable to the first container port and the thirdcontainer port, the second sterile conduit further comprising a valve.3. The apparatus of claim 1, wherein the first sterile conduit isadapted to an in-line filter having a median pore diameter small enoughto substantially prevent the flow of bacteria through the in-linefilter.
 4. The apparatus of claim 1, wherein the first container, secondcontainer, the third container, the first container and the secondcontainer, the first container and the third container, the secondcontainer and the third container, or the first container, the secondcontainer, and the third container are blood bags.
 5. The apparatus ofclaim 1, wherein the blood cells further comprise a population ofplatelets obtained from individual random donor blood, pooled randomdonor blood, or single donor blood.
 6. The apparatus of claim 1, whereinthe second container port has a frangible barrier.
 7. The apparatus ofclaim 1, wherein the first conduit or the second conduit reversiblyattaches to the first container port, the second container port, or thethird container port through a sterile dock.
 8. An apparatus forprocessing a sample of platelets, the apparatus comprising a firststerile container having one or more ports and containing a blood cellmodifying agent which comprises cytidine5′-monophospho-N-acetylneuraminic acid, and an array comprising a firstconduit and a plurality of sterile docks, wherein each of the steriledocks is reversible adaptable to blood storage containers, the bloodstorage containers having a sample of blood cells comprising plateletsand further comprising at least one port for connecting to the steriledocks of the array, wherein the platelets are introduced into the firststerile container through the conduit and are rendered cold storagecompetent by contacting the platelets with the blood cell modifyingagent present in the sterile first container.
 9. The apparatus of claim8, wherein the blood cells further comprise a population of plateletsobtained from individual random donor blood, pooled random donor blood,or single donor blood.
 10. The apparatus of claim 8, further comprisinga second container having one or more ports and containing a blood cellmodifying agent that modifies the platelet cell surface, the firstcontainer adapted to the second container through a second sterileconduit reversibly attachable to the first container port and the secondcontainer port.
 11. The apparatus of claim 10, wherein the secondsterile conduit is adapted to an in-line filter having a median porediameter small enough to substantially prevent the flow of bacteriathrough the in-line filter.
 12. The apparatus of claim 11, wherein thesecond container port has a frangible barrier.
 13. An apparatus forprocessing a sample of platelets, the apparatus comprising a firststerile container having one or more ports, the first container furthercomprising a subcontainer disposed therein, the subcontainer having aport and a frangible barrier and containing a blood cell modifying agentwhich comprises cytidine 5′-monophospho-N-acetylneuraminic acid, and anarray comprising a conduit and a plurality of sterile docks, whereineach of the sterile docks is reversible adaptable to blood storagecontainers, the blood storage containers having a sample of blood cellscomprising platelets and further comprising at least one port forconnecting to the sterile docks of the array, wherein the platelets areintroduced into the first sterile container through the conduit and arerendered cold storage competent after the platelets are contacted withthe blood cell modifying agent present in the first sterile container.14. The apparatus of claim 13, wherein the blood cells further comprisea population of platelets obtained from individual random donor blood,pooled random donor blood, or single donor blood.
 15. A method fortreating a platelet using an apparatus, wherein the apparatus isselected from the group consisting of: a. an apparatus comprising afirst sterile container having one or more ports including a firstcontainer port and containing a preparation of blood cells comprisingplatelets, a second sterile container having one or more ports includinga second container port and containing a blood cell modifying agent, thefirst container being adapted to the second container through a firststerile conduit reversibly attachable to the first container port andthe second container port, the first sterile conduit further comprisinga valve; b. an apparatus comprising a first sterile container having oneor more ports and containing a blood cell modifying agent, and an arraycomprising a first conduit and a plurality of sterile docks, whereineach of the sterile docks is reversible adaptable to blood storagecontainers, the blood storage containers having a sample of blood cellscomprising platelets and further comprising at least one port forconnecting to the sterile docks of the array; and c. an apparatuscomprising a first sterile container having one or more ports, the firstcontainer further comprising a subcontainer disposed therein, thesubcontainer having a port and a frangible barrier and containing ablood cell modifying agent, and an array comprising a conduit and aplurality of sterile docks, wherein each of the sterile docks isreversible adaptable to blood storage containers, the blood storagecontainers having a sample of blood cells comprising platelets andfurther comprising at least one port for connecting to the sterile docksof the array; wherein the steps of the method comprise contacting theplatelets with the one or more blood cell modifying agents, wherein theone or more blood cell modifying agents comprise cytidine5′-monophospho-N-acetylneuraminic acid or both UDP-galactose andcytidine 5′-monophospho-N-acetylneuraminic acid, to thereby obtaintreated platelets.
 16. The method of claim 15, wherein the conduitfurther comprises a valve, and wherein the platelets, after beingprocessed using the apparatus such that the platelets are contacted withthe one or more blood cell modifying agents, are rendered cold-storagecompetent.
 17. The method of claim 15, wherein the blood cells arecontacted with the one or more blood cell modifying agents beforeinfusion of the treated blood cells into a patient.
 18. The method ofclaim 15, wherein the blood cells are contacted with the one or moreblood cell modifying agents before cold storage of the blood cells. 19.The method of claim 15, wherein the blood cells are contacted with theone or more blood cell modifying agents at the time of blood collectionfrom a blood donor.
 20. The method of claim 15, further includingseparating the blood cells into subpopulations of platelets, plasma, redblood cells, and white blood cells.
 21. The method of claim 15, whereinthe blood cells are contacted with the one or more blood cell modifyingagents after the blood cells have been separated by apheresis.
 22. Themethod of claim 15, wherein an amount of each of the one or more bloodcell modifying agents is between about 1 micromolar and about 10millimolar.
 23. The method of claim 15, further comprising storing thetreated platelets for a period of time ranging between about 24 hoursand about 20 days.
 24. The method of claim 15, further comprisingstoring the treated platelets at a temperature ranging between about 0°C. and about 4° C.
 25. A treated blood cell obtained through a methodfor treating a platelet using an apparatus, wherein the apparatus isselected from the group consisting of: a. an apparatus comprising afirst sterile container having one or more ports including a firstcontainer port and containing a preparation of blood cells comprisingplatelets, a second sterile container having one or more ports includinga second container port and containing a blood cell modifying agent, thefirst container being adapted to the second container through a firststerile conduit reversibly attachable to the first container port andthe second container port, the first sterile conduit further comprisinga valve; b. an apparatus comprising a first sterile container having oneor more ports and containing a blood cell modifying agent, and an arraycomprising a first conduit and a plurality of sterile docks, whereineach of the sterile docks is reversible adaptable to blood storagecontainers, the blood storage containers having a sample of blood cellscomprising platelets and further comprising at least one port forconnecting to the sterile docks of the array; and c. an apparatuscomprising a first sterile container having one or more ports, the firstcontainer further comprising a subcontainer disposed therein, thesubcontainer having a port and a frangible barrier and containing ablood cell modifying agent, and an array comprising a conduit and aplurality of sterile docks, wherein each of the sterile docks isreversible adaptable to blood storage containers, the blood storagecontainers having a sample of blood cells comprising platelets andfurther comprising at least one port for connecting to the sterile docksof the array; wherein the steps of the method comprise processing theplatelets using the apparatus such that the platelets are exposed to thetwo or more blood cell modifying agents that include cytidine5′-monophospho-N-acetylneuraminic acid or both UDP-galactose andcytidine 5′-monophospho-N-acetylneuraminic acid.
 26. The treated bloodcell of claim 25, wherein the blood cells, following cold storage, aresuitable for transfusion into a patient.